Compounds and methods for peptide ribonucleic acid condensate particles for rna therapeutics

ABSTRACT

Compounds comprising condensed particles having diameters less than 1000 nm, wherein the particles comprise one or more double stranded ribonucleic acids (dsKNAs) and one or more peptides. The compounds, compositions and methods are useful for modulating gene expression by RNA Interference.

FIELD OF THE INVENTION

This invention relates generally to the fields of RNA Interference, anddelivery of RNA therapeutics. More particularly, this invention relatesto compounds and compositions of peptide ribonucleic acid condensateparticles, and their uses for medicaments and for delivery astherapeutics. This invention relates generally to methods of usingpeptide ribonucleic acid condensate compounds in RNA Interference forgene-specific inhibition of gene expression in mammals.

BACKGROUND OF THE INVENTION

RNA Interference (RNAi) refers to methods of sequence-specificpost-transcriptional gene silencing which is mediated by adouble-stranded RNA (dsRNA) called a short interfering RNA (siRNA). SeeFire, et al., Nature 391:806, 1998, and Hamilton, et al., Science286:950-951, 1999. RNAi is shared by diverse flora and phyla and isbelieved to be an evolutionarily-conserved cellular defense mechanismagainst the expression of foreign genes. See Fire, et al., Trends Genet.15:358, 1999.

RNAi is therefore a ubiquitous, endogenous mechanism that uses smallnoncoding RNAs to silence gene expression. See Dykxhoorn, D. M. and J.Lieberman, Annu. Rev. Biomed. Eng. 8:377-402, 2006. RNAi can regulateimportant genes involved in cell death, differentiation, anddevelopment. RNAi may also protect the genome from invading geneticelements, encoded by transposons and viruses. When a siRNA is introducedinto a cell, it binds to the endogenous RNAi machinery to disrupt theexpression of mRNA containing complementary sequences with highspecificity. Any disease-causing gene and any cell type or tissue canpotentially be targeted. This technique has been rapidly utilized forgene-function analysis and drug-target discovery and validation.Harnessing RNAi also holds great promise for therapy, althoughintroducing siRNAs into cells in vivo remains an important obstacle.

The mechanism of RNAi, although not yet fully characterized, is throughcleavage of a target mRNA. The RNAi response involves an endonucleasecomplex known as the RNA-induced silencing complex (RISC), whichmediates cleavage of a single-stranded RNA complementary to theantisense strand of the siRNA duplex. Cleavage of the target RNA takesplace in the middle of the region complementary to the antisense strandof the siRNA duplex (Elbashir, et al., Genes Dev. 15:188, 2001).

One way to carry out RNAi is to introduce or express a siRNA in cells.Another way is to make use of an endogenous ribonuclease III enzymecalled dicer. One activity of dicer is to process a long dsRNA intosiRNAs. See Hamilton, et al., Science 286:950-951, 1999; Berstein, etal., Nature 409:363, 2001. A siRNA derived from dicer is typically about21-23 nucleotides in overall length with about 19 base pairs duplexed.See Hamilton, et al., supra; Elbashir, et al., Genes Dev. 15:188, 2001.In essence, a long dsRNA can be introduced in a cell as a precursor of asiRNA.

The development of RNAi therapy, antisense therapy, and gene therapy,among others, has created a need for effective means of introducingactive nucleic acid-based agents into cells. In general, nucleic acidsare stable for only very limited times in cells or plasma. However,nucleic acid-based agents can be stabilized by aggregation and bindinginto condensed compounds which may exhibit particles small enough forcellular delivery.

What is need are compounds comprised of small particles which contain anactive nucleic acid agent for intracellular delivery and, ultimately, asa therapeutic, and methods for making such compounds. In particular,there is a need for compounds and methods to deliver double-stranded RNAto cells to produce the response of RNAi.

SUMMARY OF THE INVENTION

This invention overcomes these and other drawbacks in the field byproviding a range of peptide-ribonucleic acid compounds and compositionsfor use in RNA Interference and other therapeutic methods. Thisinvention particularly provides compounds and methods of makingcompounds comprising one or more ribonucleic acid agents condensed withone or more peptides into small stable particles which are active toinhibit expression of targeted genes through RNA Interference. Thissummary, taken along with the description of drawings, detaileddescription of the invention, as well as the appended examples, claims,and drawings, as a whole, encompasses the invention disclosed.

In some aspects, this invention provides a range of peptide-RNAcompounds and compositions for use in RNA Interference and othertherapeutic methods, including compounds containing RNAs and peptidescondensed into small, stable particles, which are active to inhibitexpression of targeted genes through RNAi. The compounds of thisinvention are generally provided as a wide range of admixtures orcondensates of synthetic peptides with nucleic acids.

In other aspects, the condensate compounds and compositions of thisinvention include small, stable particles of a peptide-RNA complex. Insome embodiments, these compounds and particles can be furtherstabilized by crosslinking. In other embodiments, the compounds andcompositions of this invention include a stealthing or surface modifyingagent such as polyethylene glycol to enhance delivery.

In further aspects, the compounds of this invention include condensatecomplexes of one or more ribonucleic acids and one or more peptidecomponents. The peptide components can have sufficient positive chargeto bind to a ribonucleic acid to form a non-covalently linkedpeptide-ribonucleic acid condensate compound.

In some aspects, condensate compounds of this invention may form uniformparticles. In some embodiments, the diameters of spherical particles ofpeptide-nucleic acid compounds may have a narrow distribution with anaverage of less than 1000 nanometers (nm).

The peptide-nucleic acid condensate compounds of this invention canprovide their own multicomponent formulations. In some embodiments, acompound can be combined with other agents for drug delivery such ascarriers or vehicles for delivery to a cell, or various deliverymatrices, for in vivo therapeutics.

In some embodiments, compounds are provided from one or more ribonucleicacids and one or more peptides by dissolving at least one ribonucleicacid agent in an aqueous solution, then adding at least one peptidecomponent to the aqueous solution thereby condensing particles havingdiameters less than 1000 nm, thereafter adding a second or successivepeptide components to the aqueous solution, which adds mass to theparticles.

In further embodiments, compounds are provided from one or moreribonucleic acid agents and one or more peptide components by dissolvinga first peptide component in an aqueous solution, then adding theribonucleic acid agent to the aqueous solution thereby condensingparticles having diameters less than 1000 nm, thereafter adding a secondor successive peptide components to the aqueous solution, which addsmass to the particles.

In one aspect of this invention, a peptide component is selected by itsrelative affinity for a nucleic acid. The peptide components can beselected to allow a variation of the degree of binding of the peptidecomponents to the nucleic acids.

In some aspects, ribonucleic acid-peptide condensate compounds can bereversibly-bound. Compounds of ribonucleic acids and an amount ofpositively-charged ribonucleic acid-binding peptides can besubstantially stable in an extracellular biological environment andrelease ribonucleic acids upon contact with an intracellular endosome.The release may produce the response of RNAi.

In further aspects, structures and methods of stabilizing apeptide-ribonucleic acid compound are provided including crosslinkingribonucleic acid-binding peptides within the compound. Methods ofprotecting a peptide-ribonucleic acid compound from degradation within abiological organism include crosslinking at least a portion of thepeptides within the compound.

This invention further provides uses of the compounds as medicaments andin the manufacture of medicaments for use in RNAi therapy in animals andhumans.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Diameters of condensate particles of siRNA G1498 and peptidePN183 at various concentrations of G1498 and various nitrogen tophosphorous ratios (N/P). For each group of three bars at a particularN/P, the concentration of G1498 for the leftmost bar was 100 ug/ml, forthe middle bar was 50 ug/ml, and for the rightmost bar was 10 ug/ml. AtN/P of 0.2 and 0.5, the particles were very small when the concentrationof G1498 was 10 ug/ml.

FIG. 2: Diameters of condensate particles of siRNA G1498 and peptidePN183 at various nitrogen to phosphorous ratios (N/P). For each group oftwo bars at a particular N/P, the left bar was with vortexing, while theright bar was without vortexing. Data obtained immediately after mixing.

FIG. 3: Diameters of condensate particles of siRNA G1498 and peptidePN183 at various nitrogen to phosphorous ratios (N/P). For each group oftwo bars at a particular N/P, the left bar was with vortexing, while theright bar was without vortexing. Data obtained 30 minutes after mixing.

FIG. 4: Diameters of condensate particles of siRNA G1498 and peptidePN183 at various nitrogen to phosphorous ratios (N/P). For each group oftwo bars at a particular N/P, the left bar was with vortexing, while theright bar was without vortexing. Data obtained 60 minutes after mixing.

FIG. 5: Diameters of condensate particles of siRNA G1498 and peptidePN183 at various nitrogen to phosphorous ratios (N/P). For each group oftwo bars at a particular N/P, the left bar was with vortexing, while theright bar was without vortexing. Data obtained 24 hours after mixing.

FIG. 6: Diameters of condensate particles of siRNA G1498 and peptidePN183 obtained at a concentration of G1498 of 100 ug/ml for variousvalues of pH.

FIG. 7: Diameters of condensate particles of siRNA G1498 and peptidePN183 obtained as the concentration of sodium chloride was increased.

FIG. 8: Diameters of condensate particles of siRNA G1498 and peptidePN183 obtained at various N/P ratios and various order of addition ofthe components. For each group of two bars at a particular N/P, the leftbar was obtained by adding siRNA first, while the right bar was obtainedby adding the peptide first.

FIG. 9: Transmission electron micrograph of condensate particles ofsiRNA G1498 and peptide PN183. Length legend marker is 200 nm.

FIG. 10: Transmission electron micrograph of condensate particles ofsiRNA G1498 and peptide PN183. Length legend marker is 200 nm.

FIG. 11: Knockdown assay of LPS-induced TFN-α expression (pg/ml) in amouse model by intranasal administration of a composition includingcondensate particles of siRNA Inm-4 and peptides PN183 and PN939. Buffercontrol is the leftmost bar, followed by data for condensateInm-4/PN183/PN939, followed on the right by data for compoundInm-4/PN183/PN939 crosslinked with glutaraldehyde (G). Placebo does notcontain the siRNA, and Qneg contains a non-active-siRNA.

FIG. 12: Knockdown in vitro assay of lac-z expression in rat gliosarcomafibroblast cells 9L/LacZ for condensates of the lac-z siRNA with peptidePN183 and various second peptides. Comparative data using HiPerFect™(Qiagen; Valencia, Calif.) is the leftmost bar, followed by data forvarious compounds of this invention. The N/P ratio for PN183 was 0.75,while the N/P ratio for the second peptide was 0.3.

DETAILED DESCRIPTION OF INVENTION

This invention provides a range of peptide-RNA compounds andcompositions for use in RNA Interference and other therapeutic methods.More particularly, this invention includes compounds containing RNA andpeptide condensed into small, stable particles, which are active toinhibit expression of targeted genes through RNAi.

The compounds of this invention are generally provided as admixtures orcondensates of synthetic peptides with nucleic acids. A wide range ofpeptides may be used to form the compounds. The mass of a peptide istypically less than about 120 kDa, or less than about 60 kDa, or lessthan about 30 kDa. A peptide of the compound may be a mucosalpermeability modulator or mucosal permeation enhancer.

The condensate compounds include small, stable particles of apeptide-RNA complex. These compounds and particles can be furtherstabilized by crosslinking with various reagents. In some embodiments,the compounds and compositions of this invention include a stealthing orsurface modifying agent such as polyethylene glycol to enhance delivery.

The compounds of this invention include condensate complexes comprisedof one or more ribonucleic acids and one or more peptide components. Thepeptide components can have sufficient positive charge to bind to aribonucleic acid to form a non-covalently linked peptide-ribonucleicacid condensate compound. Stable ribonucleic acid complexes are providedwhich are comprised of ribonucleic acids and an amount of ribonucleicacid-binding peptides effective to stabilize the ribonucleic acid underin vivo conditions. The binding of the components of the peptide-nucleicacid complex is due partly to ionic forces, and can involve variousother interactions such as van der Waals forces or hydrogen bonding.

Peptide-nucleic acid condensate compounds of this invention can compriseuniform particles. The diameters of spherical particles of thepeptide-nucleic acid compounds may have a narrow distribution with anaverage of less than 1000 nanometers (nm). The diameters of sphericalparticles may be less than 1000 nanometers, from about 0.5 to about 400nanometers, from about 10 to about 300 nanometers, and from about 40 toabout 100 nanometers. The magnitude of the zeta potential for stableparticles can be greater than about 20 millivolts, or greater than about30 millivolts.

As used herein, the term “uniform” means that a substantial portion ofthe particles of a compound have a narrow distribution of diameters.More than one distribution of diameters may occur in a compound ofuniform particles. A narrow distribution of diameters corresponds to apeak in the particle size distribution chart which is based on the rawcorrelation coefficient versus time data of a particle sizer instrument.Preferably, a uniform compound has at least 30% of the particles in onenarrow distribution of diameters.

The peptide-nucleic acid condensate compounds of this invention providetheir own multicomponent formulations, and can be further combined withother agents for drug delivery such as carriers or vehicles for deliveryto a cell, or various delivery matrices, for in vivo therapeutics.

The compound and compositions of this invention may be dispersed withina pharmaceutically acceptable medium, associated with a matrix, orassociated with a carrier or vehicle for delivery to a cell or subject.A solution comprised of a dispersion of the compounds or particles ofthis invention can be provided for delivery as a therapeutic.

Peptide Components

Peptide components suitable for the compounds of this invention may besynthetically or derived from natural or other sources.

The peptide components can contain from 2 to about 1000 amino acids inlength; from 2 to about 600 amino acids in length; from 2 to about 60amino acids in length; from 5 to about 30 amino acids in length; andfrom 5 to about 25 amino acids in length.

The peptide components may comprise a plurality of positive charges. Forexample, a peptide component may comprise from 1 to about 100 positivecharges, from 5 to about 30 positive charges, and from 9 to about 15positive charges. The positive charges of a peptide component can beprovided by positively-charged lysine or arginine residues.

A wide range of peptides may be used to form the peptide-nucleic acidscompounds. The mass of a peptide component is typically less than about120 kDa, or less than about 60 kDa, or less than about 30 kDa. Thepeptide of the peptide component may optionally be conjugated, orderivatized with a polymer such as a polyalkyleneoxide,polyethyleneoxide, polypropyleneoxide, or combinations thereof. Forexample, the peptide components of the compounds of this invention maybe covalently derivatized with polyethyleneglycol (PEG).

Functional domains of the polynucleotide delivery-enhancing polypeptidesare useful for the ability to deliver siNAs into cells. These functionaldomains include membrane attachment, fusogenic and nucleotide bindingregions. Membrane attachment describes the ability of the exemplarypolynucleotide delivery-enhancing polypeptide to bind the cell membrane.The fusogenic character reflects an ability to detach from the cellmembrane and enter the cytoplasm. The membrane attachment and fusogenicdomains of the peptide are closely linked mechanistically (i.e.,peptide's ability to enter the cell) and therefore may be difficult todifferentiate experimentally. Lastly, the nucleotide binding describesthe peptide's ability to bind nucleotides.

A peptide of the compound may contain structural features which areknown to enhance delivery of a compound across a barrier, such as amucosal barrier. Examples of delivery enhancing features include variousprotein transduction domains. A peptide component can be a mucosalpermeability modulator.

Examples of protein transduction domains for polynucleotidedelivery-enhancing polypeptides of the invention include:

1. TAT protein transduction domain (PTD) (SEQ ID NO: 1) KRRQRRR; 2.Penetratin PTD (SEQ ID NO: 2) RQIKIWFQNRRMKWKK; 3. VP22 PTD (SEQ ID NO:3) DAATATRGRSAASRPTERPRAPARSASRPRRPVD; 4. Kaposi FGF signal sequences(SEQ ID NO: 4) AAVALLPAVLLALLAP, and SEQ ID NO: 5) AAVLLPVLLPVLLAAP; 5.Human β3 integrin signal sequence (SEQ ID NO: 6) VTVLALGALAGVGVG; 6.gp41 fusion sequence (SEQ ID NO: 7) GALFLGWLGAAGSTMGA; 7. Caimancrocodylus Ig(v) light chain (SEQ ID NO: 8) MGLGLHLLVLAAALQGA; 8.hCT-derived peptide (SEQ ID NO: 9) LGTYTQDFNKFHTFPQTAIGVGAP; 9.Transportan (SEQ ID NO: 10) GWTLNSAGYLLKINLKALAALAKKIL; 10. Loligomer(SEQ ID NO: 11) TPPKKKRKVEDPKKKK; 11. Arginine peptide (SEQ ID NO: 12)RRRRRRR; and 12. Amphiphilic model peptide (SEQ ID NO: 13)KLALKLALKALKAALKLA.

Examples of viral fusion peptides fusogenic domains for polynucleotidedelivery-enhancing polypeptides of this invention include:

1. Influenza HA2 (SEQ ID NO: 14) GLFGAIAGFIENGWEG; 2. Sendai F1 (SEQ IDNO: 15) FFGAVIGTIALGVATA; 3. Respiratory Syncytial virus F1 (SEQ ID NO:16) FLGFLLGVGSAIASGV; 4. HIV gp41 (SEQ ID NO: 17) GVFVLGFLGFLATAGS; and5. Ebola GP2 (SEQ ID NO: 18) GAAIGLAWIPYFGPAA.

In some embodiments, polynucleotide delivery-enhancing polypeptides areprovided that incorporate a DNA-binding domain or motif whichfacilitates polypeptide-siNA complex formation and/or enhances deliveryof siNAs within the methods and compositions of the invention. ExemplaryDNA binding domains in this context include various “zinc finger”domains as described for DNA-binding regulatory proteins and otherproteins identified in Table 1, below (see, e.g., Simpson, et al., J.Biol. Chem. 278:28011-28018, 2003).

TABLE 1 Exemplary Zinc Finger Motifs of Different DNA-Binding ProteinsC₂H₂ Zinc finger motif

Prosite pattern C-x(2,4)-C-x(12)-H-x(3)-H

In Table 1, the sequences for Sp1, Sp2, Sp3, Sp4, DrosBtd, DrosSp,CeT22C8.5, and Y4pB1A.4, are herein assigned SEQ ID NOS: 19, 20, 21, 22,23, 24, 25, and 26, respectively.

Table 1 demonstrates a conservative zinc finger motif for double strandDNA binding which is characterized by the C-x(2,4)-C-x(12)-H-x(3)-Hmotif pattern (SEQ ID NO: 27), which itself can be used to select anddesign additional polynucleotide delivery-enhancing polypeptidesaccording to the invention.

Alternative DNA binding domains useful for constructing polynucleotidedelivery-enhancing polypeptides of this invention include, for example,portions of the HIV Tat protein sequence.

In some embodiments of this invention, polynucleotide delivery-enhancingpolypeptides may be constructed by combining any of the foregoingstructural elements, domains, or motifs into a single polypeptide whichmediates enhanced delivery of siNAs into target cells. For example, aprotein transduction domain of the TAT polypeptide may be fused to theN-terminal 20 amino acids of the influenza virus hemagglutinin protein,termed HA2, to yield a polynucleotide delivery-enhancing polypeptide.

The compounds of this invention can include one or more peptidecomponents. A peptide component can have sufficient positive charge tobind a ribonucleic acid to form a non-covalently boundpeptide-ribonucleic acid condensate compound. While the binding of thecomponents of the peptide-nucleic acid complex is due partly to ionicforces, the binding can also involve various other interactions such asvan der Waals forces, hydrogen bonding, or hydrophobic interactions. Acomplex may retain aqueous interactions, or a region of high solventconcentration.

Stable peptide-ribonucleic acid complexes are provided which compriseribonucleic acids and an amount of ribonucleic acid-binding peptideseffective to stabilize the ribonucleic acid under in vivo conditions.

Some example peptides useful for compounds of this invention are shownin Table 2.

TABLE 2 Peptides Peptide Structure PN183 (SEQ ID NO: 28)NH₂-KETWWETWWTEWSQPGRKKRRQRRRPPQ-Amide PN183 (SEQ ID NO: 29) Analog 1NH₂-WWTWWWWWWWEWSQPKKKKRRRRRRPPQ-Amide PN183 (SEQ ID NO: 30) Analog 2NH₂-WWWWWWWWWWSQPKKKKKKKKKK-Amide PN183 (SEQ ID NO: 31) Analog 3NH₂-WWWWWWWWWWSQPRRRRRRRRRR-Amide PN183 (SEQ ID NO: 32) Analog 4NH₂-KWWWWWWWWWEWSQPKKKKRRRRRRKKK-Amide PN938 (SEQ ID NO: 33)NH₂-(LYS-His)₁₀-amide PN939 (SEQ ID NO: 34) PEG(2kSmall)-(Lys-His)₁₀-amide PN951 (SEQ ID NO: 35)NH2-(His)₆-Arg-Ser-Val-Cys-Arg-Gln-Ile-Lys-Ile-Cys-Arg-Arg-Arg-Gly-Gly-Cys-Tyr-Try-Lys-Cys-Thr-Xaa-Arg-Pro-Tyr-amide PN970 (SEQ ID NO:36) PEG (10kS.Mal)-(Lys)₉-Gly-Leu-Phe-Gly-Ala-Ile-Ala-Gly-Phe-Ile-Glu-Xaa-Gly-Trp-Glu-Gly-Met-Ile-Asp-Gly-amide PN826 (SEQ ID NO: 37)Ac-KGSKKAVTKAQKKEGKKRKRSRKESYSVYVYKVLKQ-amide PN861 (SEQ ID NO: 38)Ac-(Arg)₉-amide PN924 (SEQ ID NO: 39) NH2-(Lys)₂₀-amide PN859 (SEQ IDNO: 40) Ac-(Arg)₁₈-amide PN907 (SEQ ID NO: 41) PEG(10kS.Mal)-(Lys)₃₀-amide PN73 (SEQ ID NO: 42)NH2-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ- amide PN526 (SEQ ID NO: 43)PEG1-KLALKLALKALKAALKLA-amide

Further examples of peptides useful for compounds of this invention aregiven in the examples appended below.

Condensate Compounds and their Preparation

This invention provides peptide-ribonucleic acid condensate compoundswhich can be comprised of particles having diameters less than about1000 nm, from about 0.5 nm to about 400 nm; from about 10 nm to about300 nm; and from about 40 nm to about 100 nm.

The peptide components of the compounds may be from 5-95% of the mass ofthe particles, or from 45-95% of the mass of the particles.

In some embodiments of this invention, peptide-nucleic acid compoundsare provided from one or more ribonucleic acid agents and one or morepeptide components by condensing the ribonucleic acid agents with thepeptide components in an aqueous solution, thereby forming particleshaving diameters less than 1000 nm.

In general, the compounds of this invention comprise peptide-nucleicacid condensates having been formed from one or more peptides and one ormore nucleic acids. The condensates are characterized in part by thenitrogen to phosphorous ratio (N/P ratio) for the peptides in relationto the nucleic acids.

A compound of this invention may be comprised of condensed particleshaving diameters less than 1000 nm, wherein each particle comprises atleast 10 double stranded ribonucleic acid (dsRNA) molecules and at least10 peptides. As used herein, “at least 10 peptides” refers to a partialmolar quantity being 10 peptide molecules, which may be the same ordifferent in structure. Thus, “at least 10 peptides” can be a partialmolar quantity of a single peptide structure, or partial molarquantities of two or more different peptide structures.

In general, as used herein, terms such as “peptide” and “nucleic acid”and “dsRNA” and “siRNA” refer to an amount of those molecules sufficientto form a compound of this invention. In other words, in general, suchterms refer to partial molar quantities rather than individualmolecules. A “peptide” is one or more peptide molecules such as, forexample, Avagadro's number of peptide molecules. “Adding two peptides toa ribonucleic acid agent” refers to an admixture of peptides of twodifferent structures, each in partial molar quantity, to the ribonucleicacid agent.

The amount of peptide bound to the nucleic acids (NAs) in a complex orcondensate can be obtained from the amount of bound nucleic acids usingthe peptide:NA charge ratio for single molecule pairing, also called thenitrogen to phosphorous ratio (N/P ratio). The amount of free peptideremaining in solution after condensation is given by mass balance. Thus,the charge ratio N/P herein refers to the initial charge ratio N/P of asingle peptide component to a single nucleic acid agent in the initialcondensate solution.

In general, the concentration of the nucleic acid agents in the solutionis limited only by their solubility. The concentrations of the peptidecomponents of the solution are adjusted to provide a desired N/P ratio.

In some embodiments, the concentrations of the peptide components of thesolution are adjusted to provide a combined N/P ratio of about one. Whenthe N/P ratio is about one, then on the basis of ionic charge neitherthe peptide components nor the nucleic acid agents are in excess.

In some embodiments, the concentration of each peptide component of thesolution is adjusted to provide an N/P ratio of from about 0.2 to about50, from about 0.5 to about 20, from about 0.5 to about 7, or from about0.5 to about 2.5.

The pH of the solution is typically less than about 11, less than about9, and less than about 8. The solution can optionally be vortexed formixing the components.

In some embodiments, the condensate compounds are prepared by addingnucleic acid agents to a solution containing the peptide components.

In some embodiments, the solution may contain an inorganic or organicsalt. For example, the aqueous solution may contain sodium chloride at aconcentration of less than or equal to about 1 M, less than or equal toabout 0.5 M, and less than or equal to about 0.25 M.

Optionally, the peptide-nucleic acid condensate compounds of aparticular distribution of sizes can be isolated from the solution. Insome embodiments, the solution containing the peptide-nucleic acidcondensate compounds is filtered to isolate particles of various sizes.

In other embodiments, the solution containing the peptide-nucleic acidcondensate compounds is dialyzed to remove excess or unbound peptidecomponents.

In some embodiments, isolated peptide-nucleic acid particles arelyophilized.

In some embodiments of this invention, peptide-nucleic acid compoundsare provided from one or more ribonucleic acid agents and one or morepeptide components by dissolving at least one ribonucleic acid agent inan aqueous solution, then adding at least one peptide component to theaqueous solution thereby condensing particles having diameters less than1000 nm, thereafter adding a second or successive peptide components tothe aqueous solution, thereby adding mass to the particles.

In some embodiments of this invention, peptide-nucleic acid compoundsare provided from one or more ribonucleic acid agents and one or morepeptide components by dissolving a first peptide component in an aqueoussolution, then adding the ribonucleic acid agent to the aqueous solutionthereby condensing particles having diameters less than 1000 nm,thereafter adding a second or successive peptide components to theaqueous solution, thereby adding mass to the particles.

In one aspect of this invention, peptide-nucleic acid compounds areprovided in which a peptide component is selected by its relativeaffinity for the nucleic acid. For example, a relative binding analysisof various peptides to a nucleic acid is performed by measurement of thedisplacement of SYBR-gold nucleic acid binding dye by the peptide. Bycharacterizing the relative affinity of the peptide components for thenucleic acids of the compounds, the peptide components can be selectedto allow a variation of the degree of binding of the peptide componentsto the nucleic acids.

Varying the degree of binding of the peptide components to the nucleicacids allows the condensate particles to be formed with astronger-binding peptide component first, followed by a weaker-bindingpeptide component, or vice-versa, or to have multiple additions ofcomponents of variable binding strength.

In some embodiments, it is desirable to have the first peptide componentwhich is condensed with the nucleic acid agent to have a higher bindingaffinity for the nucleic acid agent than succeeding peptide components.In these embodiments the concentration of the first peptide component ofthe solution is adjusted to provide an N/P ratio of from about 0.2 toabout 7, from about 0.2 to about 2.5, or from about 0.2 to about 1. Inthese embodiments the concentrations of succeeding peptide components isadjusted to provide an NIP ratio of from about 0.2 to about 50, fromabout 0.5 to about 20, from about 0.5 to about 7, or from about 0.5 toabout 2.5.

Reversibly-bound ribonucleic acid-peptide condensate compounds compriseribonucleic acids and an amount of positively-charged ribonucleicacid-binding peptides that form a ribonucleic acid-peptide condensatethat is substantially stable in an extracellular biological environmentand that can release ribonucleic acids upon contact with anintracellular endosome.

A population of peptide-nucleic acid condensates is provided in whichthe peptides comprise an amount of positively-charged residues effectiveto bind ribonucleic acids. The ribonucleic acid-peptide condensates aresubstantially stable in an extracellular biological environment and canrelease ribonucleic acids intracellularly in a manner effective toproduce the response of RNAi.

In some aspects of this invention, reagents are used to crosslink thepeptide-RNA condensates. For example, the stability of peptide-RNAcondensates may be increased by introducing dialdehyde groups, such asglutaraldehyde, to crosslink surface amine groups on the peptides orparticles. Other examples of crosslinkers include formaldehyde,acrolein, and dithiobis(succinimidylpropionate). Crosslinked condensatecompounds may have improved resistance to metabolism by serumendonucleases.

In some embodiments, a first peptide component which is condensed withthe nucleic acid agent is crosslinked before the addition of successivepeptide components. Optionally, the condensate of a first peptidecomponent can be crosslinked after the addition of successive peptidecomponents. In some embodiments, the condensate of a first peptidecomponent is crosslinked before and after the addition of successivepeptide components.

Methods of stabilizing a peptide-ribonucleic acid compound includecrosslinking ribonucleic acid-binding peptides within the compound with,for example, a glutaraldehyde crosslinker. Methods of protecting apeptide-ribonucleic acid compound from degradation within a biologicalorganism include crosslinking at least a portion of the peptides withinthe compound using, for example, a glutaraldehyde crosslinker.

The peptide-ribonucleic acid compounds of this invention can also bestabilized by addition of surface modifying agents such as surfactants,neutral lipids, or a polyethyleneoxide. For example, polyethylene glycoladded to a solution of the condensate compounds can adhere to theparticles thereof. A nonionic polyoxyethylene-polyoxypropylene blockco-polymer may be added, for example, to stabilize the particles of thecompound.

Uses of the compounds of this invention in the manufacture ofmedicaments for use in RNAi therapy in animals and humans areencompassed herein.

Nucleic Acid Agents

Nucleic acid agents useful for this invention may be single-strandednucleic acids, double-stranded nucleic acids, modified ordegradation-resistant nucleic acids, RNA, a DNA-RNA chimera, anantisense nucleic acid, or a ribozyme.

In this context, this invention provides compounds, compositions andmethods for modulating gene expression by RNA Interference. A compoundor composition of this invention may release a ribonucleic acid agent toa cell which can produce the response of RNAi. Compounds or compositionsof this invention may release ribonucleic acid agents to a cell uponcontact with an intracellular endosome. The release of a ribonucleicacid agent intracellularly may provide inhibition of gene expression inthe cell.

Ribonucleic acid agents useful for this invention may be targeted tovarious genes. For example, a siRNA agent of this invention may have asequence that is complementary to a region of a TNF-alpha gene. In someembodiments of this invention, compounds and compositions are useful toregulate expression of tumor necrosis factor-α (TNF-α). TNF-α can belinked, for example, to inflammatory processes which occur in pulmonarydiseases, and can have anti-inflammatory effects. Blocking TNF-α bydelivery of a composition of this invention can be useful to treat orprevent the signs and/or symptoms of rheumatoid arthritis.

This invention provides compounds, compositions and methods formodulating expression and activity of TNF-α by RNA Interference.

Expression and/or activity of TNF-α can be modulated by delivering to acell, for example, the siRNA molecule Inm-4. Inm-4 is a double stranded21-nt siRNA molecule with sequence homology to the human TNF-α gene.Inm-4 has a 3′ dTdT overhang on the sense strand and a 3′ dAdT overhangon the antisense strand. The primary structure of Inm-4 is

sense 5′-CCGUCAGCCGAUUUGCUAUdTdT (SEQ ID NO: 44) antisense5′-AUAGCAAAUCGGCUGACGGdTdT (SEQ ID NO: 45)

Expression and/or activity of TNF-α can be modulated by delivering to acell, for example, the siRNA molecule LC20. LC20 is a double stranded21-nt siRNA molecule with sequence homology to the human TNF-α gene.LC20 is directed against the 3′-UTR region of human TNF-α. LC20 has 19base pairs with a 3′ dTdT overhang on the sense strand and a 3′ dAdToverhang on the antisense strand. The molecular weight of the sodiumsalt form is 14,298. The primary structure of LC20 is

sense (5′) GGGUCGGAACCCAAGCUUAdTdT (SEQ ID NO: 46) antisense (5′)UAAGCUUGGGUUCCGACCCdTdA (SEQ ID NO: 47)

A siRNA of this invention may have a sequence that is complementary to aregion of a viral gene. For example, some compositions and methods ofthis invention are useful to regulate expression of the viral genome ofan influenza.

In this context, this invention provides compositions and methods formodulating expression and infectious activity of an influenza by RNAInterference. Expression and/or activity of an influenza can bemodulated by delivering to a cell, for example, a short interfering RNAmolecule having a sequence that is complementary to a region of a RNApolymerase subunit of an influenza. For example, in Table 3 are showndouble-stranded siRNA molecules with sequence homology to an RNApolymerase subunit of an influenza.

TABLE 3 Double-Stranded siRNA Molecules Targeted to Influenza Sub- siRNAunit SEQUENCE G3789 PB2 (SEQ ID NO 48) CGGGACUCUAGCAUACUUAdTdT (SEQ IDNO 49) UAAGUAUGCUAGAGUCCCGdTdT G3807 PB2 (SEQ ID NO 50)ACUGACAGCCAGACAGCGAdTdT (SEQ ID NO 51) UCGCUGUCUGGCUGUCAGUdTdT G3817 PB2(SEQ ID NO 52) AGACAGCGACCAAAAGAAUdTdT (SEQ ID NO 53)AUUCUUUUGGUCGCUGUCUdTdT G6124 PB1 (SEQ ID NO 54) AUGAAGAUCUGUUCCACCAdTdT(SEQ ID NO 55) UGGUGGAACAGAUCUUCAUdTdT G6129 PB1 (SEQ ID NO 56)GAUCUGUUCCACCAUUGAAdTdT (SEQ ID NO 57) UUCAAUGGUGGAACAGAUCdTdT G8282 PA(SEQ ID NO 58) GCAAUUGAGGAGUGCCUGAdTdT (SEQ ID NO 59)UCAGGCACUCCUCAAUUGCdTdT G8286 PA (SEQ ID NO 60) UUGAGGAGUGCCUGAUUAAdTdT(SEQ ID NO 61) UUAAUCAGGCACUCCUCAAdTdT G1498 NP (SEQ ID NO 62)GGAUCUUAUUUCUUCGGAGdTdT (SEQ ID NO 63) CUCCGAAGAAAUAAGAUCCdTdT

A siRNA of this invention may have a sequence that is complementary to aregion of a RNA polymerase subunit of an influenza.

This invention provides compositions and methods to administer siNAsdirected against a mRNA of an influenza, which effectivelydown-regulates an influenza RNA and thereby reduces, prevents, orameliorates an influenza infection.

RNA Interference Therapeutics

In some embodiments, this invention provides compounds, compositions andmethods for inhibiting expression of a target transcript in a subject byadministering to the subject a composition containing an effectiveamount of an RNAi-inducing compound such as a short interferingoligonucleotide molecule, or a precursor thereof. RNAi uses smallinterfering RNAs (siRNAs) to target messenger RNA (mRNAs) and attenuatetranslation. A siRNA as used in this invention may be a precursor fordicer processing such as, for example, a long dsRNA processed into asiRNA. This invention provides methods of treating or preventingdiseases or conditions associated with expression of a target transcriptor activity of a peptide or protein encoded by the target transcript.

A therapeutic strategy based on RNAi can be used to treat a wide rangeof diseases by shutting down the growth or function of a virus ormicroorganism, as well as by shutting down the function of an endogenousgene product in the pathway of the disease.

In some embodiments, this invention provides novel compositions andmethods for delivery of RNAi-inducing compounds such as shortinterfering oligonucleotide molecules, and precursors thereof. Inparticular, this invention provides compositions containing anRNAi-inducing compound which is targeted to one or more transcripts of acell, tissue, and/or organ of a subject.

A siRNA can be two RNA strands having a region of complementarity about19 nucleotides in length. A siRNA optionally includes one or twosingle-stranded overhangs or loops.

A shRNA can be a single RNA strand having a region ofself-complementarity. The single RNA strand may form a hairpin structurewith a stem and loop and, optionally, one or more unpaired portions atthe 5′ and/or 3′ portion of the RNA.

The active therapeutic agent can be a chemically-modified siNA withimproved resistance to nuclease degradation in vivo, and/or improvedcellular uptake, which retains RNAi activity.

A siRNA agent of this invention may have a sequence that iscomplementary to a region of a target gene. A siRNA of this inventionmay have 29-50 base pairs, for example, a dsRNA having a sequence thatis complementary to a region of a target gene. Alternately, thedouble-stranded nucleic acid can be a dsDNA.

In some embodiments, the active agent can be a short interfering nucleicacid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA),micro-RNA, or short hairpin RNA (shRNA) that can modulate expression ofa gene product.

Comparable methods and compositions are provided that target expressionof one or more different genes associated with a particular diseasecondition in a subject, including any of a large number of genes whoseexpression is known to be aberrantly increased as a causal orcontributing factor associated with the selected disease condition.

The RNAi-inducing compound of this invention can be administered inconjunction with other known treatments for a disease condition.

In some embodiments, this invention features compositions containing asmall nucleic acid molecule, such as short interfering nucleic acid, ashort interfering RNA, a double-stranded RNA, a micro-RNA, or a shorthairpin RNA, admixed or complexed with, or conjugated to, adelivery-enhancing compound.

As used herein, the terms “short interfering nucleic acid,” “siNA,”“short interfering RNA,” “siRNA,” “short interfering nucleic acidmolecule,” “short interfering oligonucleotide molecule,” and“chemically-modified short interfering nucleic acid molecule,” refer toany nucleic acid molecule capable of inhibiting or down regulating geneexpression or viral replication, for example, by mediating RNAinterference (RNAi) or gene silencing in a sequence-specific manner.

In some embodiments, the siNA is a double-stranded polynucleotidemolecule comprising self-complementary sense and antisense regions,wherein the antisense region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence in a target ribonucleic acidmolecule for down regulating expression, or a portion thereof, and thesense region comprises a nucleotide sequence corresponding to (i.e.,which is substantially identical in sequence to) the target ribonucleicacid sequence or portion thereof.

“siNA” means a small interfering nucleic acid, for example a siRNA, thatis a short-length double-stranded nucleic acid, or optionally a longerprecursor thereof. The length of useful siNAs within this invention willin some embodiments be optimized at a length of approximately 20 to 50bp. However, there is no particular limitation to the length of usefulsiNAs, including siRNAs. For example, siNAs can initially be presentedto cells in a precursor form that is substantially different than afinal or processed form of the siNA that will exist and exert genesilencing activity upon delivery, or after delivery, to the target cell.Precursor forms of siNAs may, for example, include precursor sequenceelements that are processed, degraded, altered, or cleaved at or afterthe time of delivery to yield a siNA that is active within the cell tomediate gene silencing. In some embodiments, useful siNAs will have aprecursor length, for example, of approximately 100-200 base pairs, or50-100 base pairs, or less than about 50 base pairs, which will yield anactive, processed siNA within the target cell. In other embodiments, auseful siNA or siNA precursor will be approximately 10 to 49 bp, or 15to 35 bp, or about 21 to 30 bp in length.

In some embodiments of this invention, polynucleotide delivery-enhancingpolypeptides are used to facilitate delivery of larger nucleic acidmolecules than conventional siNAs, including large nucleic acidprecursors of siNAs. For example, the methods and compositions hereinmay be employed for enhancing delivery of larger nucleic acids thatrepresent “precursors” to desired siNAs, wherein the precursor aminoacids may be cleaved or otherwise processed before, during or afterdelivery to a target cell to form an active siNA for modulating geneexpression within the target cell.

For example, a siNA precursor polynucleotide may be selected as acircular, single-stranded polynucleotide, having two or more loopstructures and a stem comprising self-complementary sense and antisenseregions, wherein the antisense region comprises a nucleotide sequencethat is complementary to a nucleotide sequence in a target nucleic acidmolecule or a portion thereof, and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof, and wherein the circular polynucleotide can be processed eitherin vivo or in vitro to generate an active siNA molecule capable ofmediating RNAi.

siNA molecules of this invention, particularly non-precursor forms, canbe less than 30 base pairs, or about 17-19 bp, or 19-21 bp, or 21-23 bp.

siRNAs can mediate selective gene silencing in the mammalian system.Hairpin RNAs, with a short loop and 19 to 27 base pairs in the stem,also selectively silence expression of genes that are homologous to thesequence in the double-stranded stem. Mammalian cells can convert shorthairpin RNA into siRNA to mediate selective gene silencing.

RISC mediates cleavage of single stranded RNA having sequencecomplementary to the antisense strand of the siRNA duplex. Cleavage ofthe target RNA takes place within the region complementary to theantisense strand of the siRNA duplex. siRNA duplexes of 21 nucleotidesare typically most active when containing two-nucleotide 3′-overhangs.

Replacing the 3′-overhanging segments of a 21-mer siRNA duplex having2-nucleotide 3′ overhangs with deoxyribonucleotides may not have anadverse effect on RNAi activity. Replacing up to 4 nucleotides on eachend of the siRNA with deoxyribonucleotides can be tolerated whereascomplete substitution with deoxyribonucleotides may result in no RNAiactivity.

Alternatively, the siNAs can be delivered as single or multipletranscription products expressed by a polynucleotide vector encoding thesingle or multiple siNAs and directing their expression within targetcells. In these embodiments the double-stranded portion of a finaltranscription product of the siRNAs to be expressed within the targetcell can be, for example, 15 to 49 bp, 15 to 35 bp, or about 21 to 30 bylong.

In some embodiments of this invention, the double-stranded region ofsiNAs in which two strands are paired may contain bulge or mismatchedportions, or both. Double-stranded portions of siNAs in which twostrands are paired are not limited to completely paired nucleotidesegments, and may contain nonpairing portions due to, for example,mismatch (the corresponding nucleotides not being complementary), bulge(lacking in the corresponding complementary nucleotide on one strand),or overhang. Nonpairing portions can be contained to the extent thatthey do not interfere with siNA formation. In some embodiments, a“bulge” may comprise 1 to 2 nonpairing nucleotides, and thedouble-stranded region of siNAs in which two strands pair up may containfrom about 1 to 7, or about 1 to 5 bulges. In addition, “mismatch”portions contained in the double-stranded region of siNAs may be presentin numbers from about 1 to 7, or about 1 to 5. Most often in the case ofmismatches, one of the nucleotides is guanine, and the other is uracil.Such mismatching may be attributable, for example, to a mutation from Cto T, G to A, or mixtures thereof, in a corresponding DNA coding forsense RNA, but other causes are also contemplated.

The terminal structure of siNAs of this invention may be either blunt orcohesive (overhanging) as long as the siNA retains its activity tosilence expression of target genes. The cohesive (overhanging) endstructure is not limited to the 3′ overhang, but includes the 5′overhanging structure as long as it retains activity for inducing genesilencing. In addition, the number of overhanging nucleotides is notlimited to 2 or 3 nucleotides, but can be any number of nucleotides aslong as it retains activity for inducing gene silencing. For example,overhangs may comprise from 1 to about 8 nucleotides, or from 2 to 4nucleotides.

The length of siNAs having cohesive (overhanging) end structure may beexpressed in terms of the paired duplex portion and any overhangingportion at each end. For example, a 25/27-mer siNA duplex with a 2-bp 3′antisense overhang has a 25-mer sense strand and a 27-mer antisensestrand, where the paired portion has a length of 25 bp.

Any overhang sequence may have low specificity to a target gene, and maynot be complementary (antisense) or identical (sense) to the target genesequence. As long as the siNA retains activity for gene silencing, itmay contain in the overhang portion a low molecular weight structure,for example, a natural RNA molecule such as a tRNA, an rRNA, a viralRNA, or an artificial RNA molecule.

The terminal structure of the siNAs may have a stem-loop structure inwhich ends of one side of the double-stranded nucleic acid are connectedby a linker nucleic acid, e.g., a linker RNA. The length of thedouble-stranded region (stem-loop portion) can be, for example, 15 to 49bp, or 15 to 35 bp, or about 21 to 30 by long. Alternatively, the lengthof the double-stranded) region that is a final transcription product ofsiNAs to be expressed in a target cell may be, for example,approximately 15 to 49 bp, or 15 to 35 bp, or about 21 to 30 by long.

The siNA can contain a single stranded polynucleotide having anucleotide sequence complementary to a nucleotide sequence in a targetnucleic acid molecule, or a portion thereof, wherein the single strandedpolynucleotide can contain a terminal phosphate group, such as a5′-phosphate (see for example, Martinez, et al., Cell. 110:563-574,2002, and Schwarz, et al., Molecular Cell 10:537-568, 2002, or5′,3′-diphosphate.

As used herein, the term siNA molecule is not limited to moleculescontaining only naturally-occurring RNA or DNA, but also encompasseschemically-modified nucleotides and non-nucleotides. In someembodiments, the short interfering nucleic acid molecules of theinvention lack 2′-hydroxy (2′-OH) containing nucleotides. In someembodiments, short interfering nucleic acids do not require the presenceof nucleotides having a 2′-hydroxy group for mediating RNAi and as such,short interfering nucleic acid molecules of this invention optionally donot include any ribonucleotides (e.g., nucleotides having a 2′-OHgroup). siNA molecules that do not require the presence ofribonucleotides within the siNA molecule to support RNAi can, however,have an attached linker or linkers or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′-OH groups. siNA molecules can comprise ribonucleotides in at leastabout 5, 10, 20, 30, 40, or 50% of the nucleotide positions.

As used herein, the term siNA encompasses nucleic acid molecules thatare capable of mediating sequence specific RNAi such as, for example,short interfering RNA (siRNA) molecules, double-stranded RNA (dsRNA)molecules, micro-RNA molecules, short hairpin RNA (shRNA) molecules,short interfering oligonucleotide molecules, short interfering nucleicacid molecules, short interfering modified oligonucleotide molecules,chemically-modified siRNA molecules, and post-transcriptional genesilencing RNA (ptgsRNA) molecules, among others.

In some embodiments, siNA molecules comprise separate sense andantisense sequences or regions, wherein the sense and antisense regionsare covalently linked by nucleotide or non-nucleotide linker molecules,or are non-covalently linked by ionic interactions, hydrogen bonding,van der waals interactions, hydrophobic intercations, and/or stackinginteractions.

“Antisense RNA” is an RNA strand having a sequence complementary to atarget gene mRNA, that can induce RNAi by binding to the target genemRNA.

“Sense RNA” is an RNA strand having a sequence complementary to anantisense RNA, and anneals to its complementary antisense RNA to form asiRNA.

As used herein, the term “RNAi construct” or “RNAi precursor” refers toan RNAi-inducing compound such as small interfering RNAs (siRNAs),hairpin RNAs, and other RNA species which can be cleaved in vivo to forma siRNA. RNAi precursors herein also include expression vectors (alsoreferred to as RNAi expression vectors) capable of giving rise totranscripts which form dsRNAs or hairpin RNAs in cells, and/ortranscripts which can produce siRNAs in vivo.

A siHybrid molecule is a double-stranded nucleic acid that has a similarfunction to siRNA. Instead of a double-stranded RNA molecule, a siHybridis comprised of an RNA strand and a DNA strand. Preferably, the RNAstrand is the antisense strand which binds to a target mRNA. ThesiHybrid created by the hybridization of the DNA and RNA strands have ahybridized complementary portion and preferably at least one 3′overhanging end.

siNAs for use within the invention can be assembled from two separateoligonucleotides, where one strand is the sense strand and the other isthe antisense strand, wherein the antisense and sense strands areself-complementary (i.e., each strand comprises nucleotide sequence thatis complementary to nucleotide sequence in the other strand; such aswhere the antisense strand and sense strand form a duplex or doublestranded structure, for example wherein the double stranded region isabout 19 base pairs). The antisense strand may comprise a nucleotidesequence that is complementary to a nucleotide sequence in a targetnucleic acid molecule or a portion thereof, and the sense strand maycomprise a nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. Alternatively, the siNA can be assembledfrom a single oligonucleotide, where the self-complementary sense andantisense regions of the siNA are linked by means of a nucleicacid-based or non-nucleic acid-based linker(s).

In some embodiments, siNAs for intracellular delivery can be apolynucleotide with a duplex, asymmetric duplex, hairpin or asymmetrichairpin secondary structure, having self-complementary sense andantisense regions, wherein the antisense region comprises a nucleotidesequence that is complementary to a nucleotide sequence in a separatetarget nucleic acid molecule or a portion thereof, and the sense regioncomprises a nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof.

Examples of chemical modifications that can be made in an siNA includephosphorothioate internucleotide linkages, 2′-deoxyribonucleotides,2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides,“universal base” nucleotides, “acyclic” nucleotides, 5-C-methylnucleotides, and terminal glyceryl and/or inverted deoxy abasic residueincorporation.

The antisense region of a siNA molecule can include a phosphorothioateinternucleotide linkage at the 3′-end of said antisense region. Theantisense region can comprise about one to about five phosphorothioateinternucleotide linkages at the 5′-end of said antisense region. The3′-terminal nucleotide overhangs of a siNA molecule can includeribonucleotides or deoxyribonucleotides that are chemically-modified ata nucleic acid sugar, base, or backbone. The 3′-terminal nucleotideoverhangs can include one or more universal base ribonucleotides. The3′-terminal nucleotide overhangs can comprise one or more acyclicnucleotides.

For example, a chemically-modified siNA can have 1, 2, 3, 4, 5, 6, 7, 8,or more phosphorothioate internucleotide linkages in one strand, or canhave 1 to 8 or more phosphorothioate internucleotide linkages in eachstrand. The phosphorothioate internucleotide linkages can be present inone or both oligonucleotide strands of the siNA duplex, for example inthe sense strand, the antisense strand, or both strands.

siNA molecules can comprise one or more phosphorothioate internucleotidelinkages at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe sense strand, the antisense strand, or in both strands. For example,an exemplary siNA molecule can include 1, 2, 3, 4, 5, or moreconsecutive phosphorothioate internucleotide linkages at the 5′-end ofthe sense strand, the antisense strand, or both strands.

In some embodiments, a siNA molecule includes 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more pyrimidine phosphorothioate internucleotide linkages in thesense strand, the antisense strand, or in both strands.

In some embodiments, a siNA molecule includes 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more purine phosphorothioate internucleotide linkages in thesense strand, the antisense strand, or in both strands.

A siNA molecule can include a circular nucleic acid molecule, whereinthe siNA is about 38 to about 70, for example, about 38, 40, 45, 50, 55,60, 65, or 70 nucleotides in length, having about 18 to about 23, forexample, about 18, 19, 20, 21, 22, or 23 base pairs, wherein thecircular oligonucleotide forms a dumbbell-shaped structure having about19 base pairs and 2 loops.

A circular siNA molecule can contain two loop motifs, wherein one orboth loop portions of the siNA molecule is biodegradable. For example,the loop portions of a circular siNA molecule may be transformed in vivoto generate a double-stranded siNA molecule with 3′-terminal overhangs,such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

Modified nucleotides in a siNA molecule can be in the antisense strand,the sense strand, or both. For example, modified nucleotides can have aNorthern conformation (e.g., Northern pseudorotation cycle, see forexample, Saenger, Principles of Nucleic Acid Structure, Springer-Verlaged., 1984). Examples of nucleotides having a Northern configurationinclude locked nucleic acid (LNA) nucleotides (e.g., 2′-O,4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-methoxyethoxy (MOE)nucleotides, 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides,2′-deoxy-2′-chloro nucleotides, 2′-azido nucleotides, and 2′-O-methylnucleotides.

Chemically modified nucleotides can be resistant to nuclease degradationwhile at the same time maintaining the capacity to mediate RNAi.

The sense strand of a double stranded siNA molecule may have a terminalcap moiety such as an inverted deoxyabasic moiety, at the 3′-end,5′-end, or both 3′ and 5′-ends of the sense strand.

Examples of conjugates include conjugates and ligands described inVargeese, et al., U.S. application Ser. No. 10/427,160, filed Apr. 30,2003, incorporated by reference herein in its entirety, including thedrawings.

In some embodiments of this invention, the conjugate may be covalentlyattached to the chemically-modified siNA molecule via a biodegradablelinker. For example, the conjugate molecule may be attached at the3′-end of either the sense strand, the antisense strand, or both strandsof the chemically-modified siNA molecule.

In some embodiments, the conjugate molecule is attached at the 5′-end ofeither the sense strand, the antisense strand, or both strands of thechemically-modified siNA molecule. In some embodiments, the conjugatemolecule is attached both the 3′-end and 5′-end of either the sensestrand, the antisense strand, or both strands of the chemically-modifiedsiNA molecule, or any combination thereof.

In some embodiments, a conjugate molecule comprises a molecule thatfacilitates delivery of a chemically-modified siNA molecule into abiological system, such as a cell.

In some embodiments, a conjugate molecule attached to thechemically-modified siNA molecule is a polyethylene glycol, human serumalbumin, or a ligand for a cellular receptor that can mediate cellularuptake. Examples of specific conjugate molecules contemplated by theinstant invention that can be attached to chemically-modified siNAmolecules are described in Vargeese, et al., U.S. Patent Publication No.20030130186 and U.S. Patent Publication No. 20040110296, which are eachhereby incorporated by reference in their entirety.

A siNA may be contain a nucleotide, non-nucleotide, or mixednucleotide/non-nucleotide linker that joins the sense region of the siNAto the antisense region of the siNA. In some embodiments, a nucleotidelinker can be 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In someembodiments, the nucleotide linker can be a nucleic acid aptamer. Asused herein, the terms “aptamer” or “nucleic acid aptamer” encompass anucleic acid molecule that binds specifically to a target molecule,wherein the nucleic acid molecule contains a sequence that is recognizedby the target molecule in its natural setting. Alternately, an aptamercan be a nucleic acid molecule that binds to a target molecule where thetarget molecule does not naturally bind to a nucleic acid.

For example, the aptamer can be used to bind to a ligand-binding domainof a protein, thereby preventing interaction of the naturally occurringligand with the protein. See, for example, Gold, et al., Annu. Rev.Biochem. 64:763, 1995; Brody and Gold, J. Biotechnol. 74:5, 2000; Sun,Curr. Opin. Mol. Ther. 2:100, 2000; Kusser, J. Biotechnol. 74:27, 2000;Hermann and Patel, Science 287:820, 2000; and Jayasena, ClinicalChemistry 45:1628, 1999.

A non-nucleotide linker can be an abasic nucleotide, polyether,polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, orother polymeric compounds (e.g., polyethylene glycols such as thosehaving between 2 and 100 ethylene glycol units). Specific examplesinclude those described by Seela and Kaiser, Nucleic Acids Res. 18:6353,1990, and Nucleic Acids Res. 15:3113, 1987; Cload and Schepartz, J. Am.Chem. Soc. 113:6324, 1991; Richardson and Schepartz, J. Am. Chem. Soc.113:5109, 1991; Ma, et al., Nucleic Acids Res. 21:2585, 1993, andBiochemistry 32:1751, 1993; Durand, et al., Nucleic Acids Res. 18:6353,1990; McCurdy, et al., Nucleosides & Nucleotides 10:287, 1991; Jschke,et al., Tetrahedron Lett. 34:301, 1993; Ono, et al., Biochemistry30:9914, 1991; Arnold, et al., International Publication No. WO89/02439; Usman, et al., International Publication No. WO 95/06731;Dudycz, et al., International Publication No. WO 95/11910, and Ferentzand Verdine, J. Am. Chem. Soc. 113:4000, 1991.

A “non-nucleotide linker” refers to a group or compound that can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound can be abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine, for example at the C1 position of the sugar.

In some embodiments, modified siNA molecule can have phosphate backbonemodifications including one or more phosphorothioate,phosphorodithioate, methylphosphonate, phosphotriester, morpholino,amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate,sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilylsubstitutions. Examples of oligonucleotide backbone modifications aregiven in Hunziker and Leumann, Nucleic Acid Analogues: Synthesis andProperties, in Modern Synthetic Methods, VCH, pp. 331-417, 1995, andMesmaeker, et al., Novel Backbone Replacements for Oligonucleotides, inCarbohydrate Modifications in Antisense Research, ACS, pp. 24-39, 1994.

siNA molecules, which can be chemically-modified, can be synthesized by:(a) synthesis of two complementary strands of the siNA molecule; and (b)annealing the two complementary strands together under conditionssuitable to obtain a double-stranded siNA molecule. In some embodiments,synthesis of the complementary portions of the siNA molecule is by solidphase oligonucleotide synthesis, or by solid phase tandemoligonucleotide synthesis.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers, etal., Methods in Enzymology 211:3-19, 1992; Thompson, et al.,International PCT Publication No. WO 99/54459; Wincott, et al., NucleicAcids Res. 23:2677-2684, 1995; Wincott, et al., Methods Mol. Bio. 74:59,1997; Brennan, et al., Biotechnol Bioeng. 61:33-45, 1998; and Brennan,U.S. Pat. No. 6,001,311. Synthesis of RNA, including certain siNAmolecules of the invention, follows general procedures as described, forexample, in Usman, et al., J. Am. Chem. Soc. 109:7845, 1987; Scaringe,et al., Nucleic Acids Res. 18:5433, 1990; and Wincott, et al., NucleicAcids Res. 23:2677-2684, 1995; Wincott, et al., Methods Mol. Bio. 74:59,1997.

An “asymmetric hairpin” as used herein is a linear siNA moleculecomprising an antisense region, a loop portion that can comprisenucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complementary nucleotides to base pair with theantisense region and form a duplex with loop.

An “asymmetric duplex” as used herein is a siNA molecule having twoseparate strands comprising a sense region and an antisense region,wherein the sense region comprises fewer nucleotides than the antisenseregion to the extent that the sense region has enough complementarynucleotides to base pair with the antisense region and form a duplex.

To “modulate gene expression” as used herein is to upregulate ordown-regulate expression of a target gene, which can includeupregulation or downregulation of mRNA levels present in a cell, or ofmRNA translation, or of synthesis of protein or protein subunits,encoded by the target gene.

The terms “inhibit”, “down-regulate”, or “reduce expression,” as usedherein mean that the expression of the gene, or level of RNA moleculesor equivalent RNA molecules encoding one or more proteins or proteinsubunits, or level or activity of one or more proteins or proteinsubunits encoded by a target gene, is reduced below that observed in theabsence of the nucleic acid molecules (e.g., siNA) of the invention.

“Gene silencing” as used herein refers to partial or complete inhibitionof gene expression in a cell and may also be referred to as “geneknockdown.” The extent of gene silencing may be determined by methodsknown in the art, some of which are summarized in InternationalPublication No. WO 99/32619.

As used herein, the terms “ribonucleic acid” and “RNA” refer to amolecule containing at least one ribonucleotide residue. Aribonucleotide is a nucleotide with a hydroxyl group at the 2′ positionof a beta-D-ribo-furanose moiety. These terms include double-strandedRNA, single-stranded RNA, isolated RNA such as partially purified RNA,essentially pure RNA, synthetic RNA, recombinantly produced RNA, as wellas modified and altered RNA that differs from naturally occurring RNA bythe addition, deletion, substitution, modification, and/or alteration ofone or more nucleotides. Alterations of an RNA can include addition ofnon-nucleotide material, such as to the end(s) of a siNA or internally,for example at one or more nucleotides of an RNA.

Nucleotides in an RNA molecule include non-standard nucleotides, such asnon-naturally occurring nucleotides or chemically synthesizednucleotides or deoxynucleotides. These altered RNAs can be referred toas analogs.

By “highly conserved sequence region” is meant, a nucleotide sequence ofone or more regions in a target gene does not vary significantly fromone generation to the other or from one biological system to the other.

By “sense region” is meant a nucleotide sequence of a siNA moleculehaving complementarity to an antisense region of the siNA molecule. Inaddition, the sense region of a siNA molecule can comprise a nucleicacid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of a siNA moleculehaving complementarity to a target nucleic acid sequence. In addition,the antisense region of a siNA molecule can include a nucleic acidsequence having complementarity to a sense region of the siNA molecule.

By “target nucleic acid” is meant any nucleic acid sequence whoseexpression or activity is to be modulated. A target nucleic acid can beDNA or RNA.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence either by traditionalWatson-Crick or by other non-traditional modes of binding.

The term “biodegradable linker” as used herein, refers to a nucleic acidor non-nucleic acid linker molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule to a siNA molecule or the sense andantisense strands of a siNA molecule. The biodegradable linker isdesigned such that its stability can be modulated for a particularpurpose, such as delivery to a particular tissue or cell type. Thestability of a nucleic acid-based biodegradable linker molecule can bevariously modulated, for example, by combinations of ribonucleotides,deoxyribonucleotides, and chemically-modified nucleotides, such as2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl,and other 2′-modified or base modified nucleotides. The biodegradablenucleic acid linker molecule can be a dimer, trimer, tetramer or longernucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotidesin length, or can comprise a single nucleotide with a phosphorus-basedlinkage, for example, a phosphoramidate or phosphodiester linkage. Thebiodegradable nucleic acid linker molecule can also comprise nucleicacid backbone, nucleic acid sugar, or nucleic acid base modifications.

In connection with 2′-modified nucleotides as described herein, by“amino” is meant 2′-NH₂ or 2′-O—NH₂, which can be modified orunmodified. Such modified groups are described, for example, inEckstein, et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic, et al.,U.S. Pat. No. 6,248,878.

Administration

Some methods for delivery of nucleic acid molecules for use within theninvention are described, for example, in Akhtar, et al., Trends CellBio. 2:139, 1992; Delivery Strategies for Antisense OligonucleotideTherapeutics, ed. Akhtar, 1995; Maurer, et al., Mol. Membr. Biol.16:129-140, 1999; Hofland and Huang, Handb. Exp. Pharmacol. 137:165-192,1999; and Lee, et al., ACS Symp. Ser. 752:184-192, 2000. Sullivan, etal., International PCT Publication No. WO 94/02595, further describesgeneral methods for delivery of enzymatic nucleic acid molecules. Theseprotocols can be utilized to supplement or complement delivery ofvirtually any nucleic acid molecule contemplated within the invention.

Nucleic acid molecules and peptides can be administered to cells by avariety of methods known to those of skill in the art, including, butnot restricted to, administration within formulations that comprise thesiNA and peptide alone, or that further comprise one or more additionalcomponents, such as a pharmaceutically acceptable carrier, diluent,excipient, adjuvant, emulsifier, buffer, stabilizer, preservative, andthe like. In certain embodiments, the siNA and/or the peptide can beencapsulated in liposomes, administered by iontophoresis, orincorporated into other vehicles, such as hydrogels, cyclodextrins,biodegradable nanocapsules, bioadhesive microspheres, or proteinaceousvectors (see e.g., O'Hare and Normand, International PCT Publication No.WO 00/53722). Alternatively, a nucleic acid/peptide/vehicle combinationcan be locally delivered by direct injection or by use of an infusionpump. Direct injection of the nucleic acid molecules of the invention,whether subcutaneous, intramuscular, or intradermal, can take placeusing standard needle and syringe methodologies, or by needle-freetechnologies such as those described in Conry et al., Clin. Cancer Res.5:2330-2337, 1999, and Barry, et al., International PCT Publication No:WO 99/31262.

The compositions of the instant invention can be effectively employed aspharmaceutical agents. Pharmaceutical agents prevent, modulate theoccurrence or severity of, or treat (alleviate one or more symptom(s) toa detectable or measurable extent) of a disease state or other adversecondition in a patient.

Thus within additional embodiments the invention provides pharmaceuticalcompositions and methods featuring the presence or administration of oneor more polynucleic acid(s), typically one or more siNAs, combined,complexed, or conjugated with a peptide, optionally formulated with apharmaceutically-acceptable carrier, such as a diluent, stabilizer,buffer, and the like.

The present invention satisfies additional objects and advantages byproviding short interfering nucleic acid (siNA) molecules that modulateexpression of genes associated with a particular disease state or otheradverse condition in a subject. Typically, the siNA will target a genethat is expressed at an elevated level as a causal or contributingfactor associated with the subject disease state or adverse condition.In this context, the siNA will effectively downregulate expression ofthe gene to levels that prevent, alleviate, or reduce the severity orrecurrence of one or more associated disease symptoms. Alternatively,for various distinct disease models where expression of the target geneis not necessarily elevated as a consequence or sequel of disease orother adverse condition, down regulation of the target gene willnonetheless result in a therapeutic result by lowering gene expression(i.e., to reduce levels of a selected mRNA and/or protein product of thetarget gene). Alternatively, siNAs of the invention may be targeted tolower expression of one gene, which can result in upregulation of a“downstream” gene whose expression is negatively regulated by a productor activity of the target gene.

This siNAs of the present invention may be administered in any form, forexample transdermally or by local injection. Comparable methods andcompositions are provided that target expression of one or moredifferent genes associated with a selected disease condition in animalsubjects, including any of a large number of genes whose expression isknown to be aberrantly increased as a causal or contributing factorassociated with the selected disease condition.

Negatively charged polynucleotides of the invention (e.g., RNA or DNA)can be administered to a patient by any standard means, with or withoutstabilizers, buffers, and the like, to form a pharmaceuticalcomposition. When it is desired to use a liposome delivery mechanism,standard protocols for formation of liposomes can be followed. Thecompositions of the present invention may also be formulated and used astablets, capsules or elixirs for oral administration, suppositories forrectal administration, sterile solutions, suspensions for injectableadministration, and the other compositions known in the art.

The present invention also includes pharmaceutically acceptableformulations of the compositions described herein. These formulationsinclude salts of the above compounds, e.g., acid addition salts, forexample, salts of hydrochloric, hydrobromic, acetic acid, and benzenesulfonic acid.

The siNAs can also be administered in the form of suppositories, e.g.,for rectal administration of the drug. These compositions can beprepared by mixing the drug with a suitable non-irritating excipientthat is solid at ordinary temperatures but liquid at the rectaltemperature and will therefore melt in the rectum to release the drug.Such materials include cocoa butter and polyethylene glycols.

Nucleic acid molecules can be administered to cells by a variety ofmethods known to those of skill in the art, including, but notrestricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as biodegradable polymers,hydrogels, cyclodextrins (see for example, Gonzalez, et al.,Bioconjugate Chem. 10:1068-1074, 1999; Wang, et al., International PCTPublication Nos. WO 03/47518 and WO 03/46185), poly(lactic-co-glycolicacid) (PLGA) and PLCA microspheres (see for example, U.S. Pat. No.6,447,796 and U.S. Patent Application Publication No. US 2002130430),biodegradable nanocapsules, and bioadhesive microspheres, or byproteinaceous vectors (O'Hare and Normand, International PCT PublicationNo. WO 00/53722). Alternatively, the nucleic acid/vehicle combination islocally delivered by direct injection or by use of an infusion pump.Direct injection of the nucleic acid molecules of the invention, whethersubcutaneous, intramuscular, or intradermal, can take place usingstandard needle and syringe methodologies, or by needle-freetechnologies such as those described in Conry, et al., Clin. Cancer Res.5:2330-2337, 1999, and Barry, et al., International PCT Publication No.WO 99/31262. The molecules of the instant invention can be used aspharmaceutical agents. Pharmaceutical agents prevent, modulate theoccurrence, or treat (alleviate a symptom to some extent, preferably allof the symptoms) of a disease state in a subject.

Any one or combination of the cationic peptides of the present inventionmay be selected or combined to yield effective polynucleotidedelivery-enhancing polypeptide reagents to induce or facilitateintracellular delivery of siNAs within the methods and compositions ofthe invention.

Pharmaceutical Composition

The present invention also includes pharmaceutically acceptableformulations or compositions of the compounds described herein. Theseformulations include organic and inorganic salts of the above compounds,e.g., acid addition salts, for example, salts of hydrochloric,hydrobromic, acetic acid, and benzene sulfonic acid.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Additional excipients, for example sweetening,flavoring and coloring agents, can also be present.

Pharmaceutical compositions of this invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil, or amineral oil, or mixtures thereof. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

The pharmaceutical compositions can be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension can beformulated using a suitable dispersing or wetting agent, and/or asuspending agent. A sterile injectable preparation can also be a sterileinjectable solution or suspension in a non-toxic parentally acceptablediluent or solvent, for example as a solution in 1,3-butanediol.

Among the acceptable carriers, vehicles and solvents for apharmaceutical composition that can be employed are water, Ringer'ssolution, and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a carrier, vehicle, solvent,or suspending medium. For this purpose, any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid find use in the preparation of injectables.

All publications, references, patents, and patent applications citedherein are each hereby specifically incorporated by reference in theirentirety.

While this invention has been described in relation to certainembodiments; and many details have been set forth for purposes ofillustration, it will be apparent to those skilled in the art that thisinvention includes additional embodiments, and that some of the detailsdescribed herein may be varied considerably without departing from thisinvention. This invention includes such additional embodiments,modifications and equivalents.

The use herein of the terms “a,” “an,” “the,” and similar terms indescribing the invention, and in the claims, are to be construed toinclude both the singular and the plural. The terms “comprising,”“having,” “including,” and “containing” are to be construed asopen-ended terms which mean, for example, “including, but not limitedto.” Recitation of a range of values herein is intended to referindividually to each separate value falling within the range as if itwere individually recited herein, whether or not some of the valueswithin the range are expressly recited. Specific values employed hereinwill be understood as exemplary and not to limit the scope of theinvention.

The examples given herein, and the exemplary language used herein aresolely for the purpose of illustration, and are not intended to limitthe scope of the invention.

EXAMPLES Preparation Example 1

PN0826:siRNA compounds in water. A compound was prepared by: Adding82.12 μl of RNase free water to a centrifuge tube, and then 10 μl ofG1498 (1 mg/ml, in RNase free water). The solution was vortexed to mix.Finally, 7.88 μl of PN0826 was added (1 mg/ml, in RNase free water) andvortexed to mix.

Preparation Example 2

PN0826, F-108 and water. A compound was prepared by: in a centrifugetube, 82.12 μl RNase free water was added first and then 10 μl of G1498(1 mg/ml, in RNase free water). Vortexed to mix. Then added 7.88 μl ofPN0826 (1 mg/ml, in RNase free water) and vortexed to mix. Finally added5 μl Pluronic F108 (20 mg/ml, 0.2 μM filtered) and pipetted to mix.

Preparation Example 3

Cy5-Inm4, PN0183 and overnight. A compound was prepared by: in acentrifuge tube, 119.40 μl of 10 mM Hepes/5% dextrose buffer (pH 5.0)was added first, then 15.60 μl of peptide PN0183 (2 mg/ml, in RNase freewater), and vortexed to mix. The solution was stored at 4° overnight.Finally, 15 μl Cy5-Inm4 (1 mg/ml, in RNase free water) was added andvortexed again to mix.

Preparation Example 4

Cy5-Inm4, PN0183, F127 and overnight. A compound was prepared by: in acentrifuge tube, 119.40 μl of 10 mM Hepes/5% dextrose buffer (pH 5.0)was added first, then 15.60 μl of peptide PN0183 (2 mg/ml, in RNase freewater) and 7.5 μl Pluronic F127 (20 mg/ml, 0.2 μM filtered). Vortexed tomix. The solution was stored at 4° overnight. Finally, 15 μl Cy5-Inm4 (1mg/ml, in RNase free water) was added and vortexed again to mix.

Preparation Example 5

G1498, PN0183, water for dilution and peptide first. A compound wasprepared by: in a centrifuge tube, 85.83 μl of 10 mM Hepes/5% dextrosebuffer (pH5.0) was added first, then 4.17 μl of peptide PN0183 (5 mg/ml,in RNase free water), and vortexed to mix. Finally, 10 μl G1498 (1mg/ml, in RNase free water) was added to the solution and vortexed againto mix.

Preparation Example 6

G1498, PN0183, buffer for dilution and peptide first. A compound wasprepared by: in a centrifuge tube, 85.83 μl of 10 mM Hepes/5% dextrosebuffer (pH 5.0) was added first, then 4.17 μl of peptide PN0183 (5mg/ml, in 10 mM Hepes/5% dextrose buffer of pH 5.0), and vortexed tomix. Finally, 10 μl G1498 (1 mg/ml, in 10 mM Hepes/5% dextrose buffer ofpH 5.0) was added to the solution and vortexed again to mix.

Preparation Example 7

G1498, PN0183, peptide first and without vortexing. A compound wasprepared by: in a centrifuge tube, 85.83 μl of 10 mM Hepes/5% dextrosebuffer (pH5.0) was added first, then 4.17 μl of peptide PN0183 (5 mg/ml,in 10 mM Hepes/5% dextrose buffer of pH 5.0), and pipetted to mix.Finally, 10 μl G1498 (1 mg/ml, in 10 mM Hepes/5% dextrose buffer of pH5.0) was added to the solution and pipetted again to mix.

Preparation Example 8

G1498, PN0183, peptide first and lower concentration by diluting down. Acompound was prepared by: in a centrifuge tube, 85.83 μl of 10 mMHepes/5% dextrose buffer (pH 5.0) was added first, then 4.17 μl ofpeptide PN0183 (5 mg/ml, in 10 mM Hepes/5% dextrose buffer of pH 5.0),and vortexed to mix. 10 μl G1498 (1 mg/ml, in 10 mM Hepes/5% dextrosebuffer of pH 5.0) was added to the solution and vortexed again to mix.Finally, the solution was diluted 10 times to lower concentration.

Preparation Example 9

G1498, PN0183 and siRNA first. A compound was prepared by: in acentrifuge tube, 85.83 μl of 10 mM Hepes/5% dextrose buffer (pH 5.0) wasadded first, then 10 μl G1498 (1 mg/ml, in 10 mM Hepes/5% dextrosebuffer of pH 5.0) and vortexed to mix.

Preparation Example 10

G1498, PN0183, peptide first and wait for 30 minutes. A compound wasprepared by: in a centrifuge tube, 85.83 μl of 10 mM Hepes/5% dextrosebuffer (pH 5.0) was added first, then 4.17 μl of peptide PN0183 (5mg/ml, in 10 mM Hepes/5% dextrose buffer of pH 5.0), and vortexed tomix. Finally, 10 μl G1498 (1 mg/ml, in 10 M Hepes/5% dextrose buffer ofpH 5.0) was added to the solution and vortexed again to mix. Thesolution was equilibrated on ice for 30 minutes.

Preparation Example 11

G1498, PN0183, peptide first and wait for 60 minutes. A compound wasprepared by: in a centrifuge tube, 85.83 μl of 10 mM Hepes/5% dextrosebuffer (pH 5.0) was added first, then 4.17 μl of peptide PN0183 (5mg/ml, in 10 mM Hepes/5% dextrose buffer of pH 5.0), and vortexed tomix. Finally, 10 μl G1498 (1 mg/ml, in 10 mM Hepes/5% dextrose buffer ofpH 5.0) was added to the solution and vortexed again to mix. Thesolution was equilibrated on ice for 60 minutes.

Preparation Example 12

G1498, PN0183, peptide first and wait for 24 hrs. A compound wasprepared by: in a centrifuge tube, 85.83 μl of 10 mM Hepes/5% dextrosebuffer (pH 5.0) was added first, then 4.17 μl of peptide PN0183 (5mg/ml, in 10 mM Hepes/5% dextrose buffer of pH 5.0), and vortexed tomix. Finally, 10 μl G1498 (1 mg/ml, in 10 mM Hepes/5% dextrose buffer ofpH 5.0) was added to the solution and vortexed again to mix. Thesolution was equilibrated on ice for 24 hrs.

Preparation Example 13

Inm4, PN0183, PN0939 and siRNA added right before dosing. A compound wasprepared by: in a centrifuge tube, 259.1 μl of 10 mM Hepes/5% dextrosebuffer (pH 5.0) was added first, then 15.60 μl of PN0183 (5 mg/ml, in 10mM Hepes/5% dextrose buffer of pH 5.0) and 10.30 μl of PN0939 (5 mg/ml,in 10 mM Hepes/5% dextrose buffer of pH 5.0). Vortexed to mix. Finally15.00 μl of Inm4 (5 mg/ml, 10 mM Hepes/5% dextrose buffer of pH 5.0) wasadded. Vortexed to mix.

Preparation Example 14

Inm4, order of siRNA, PN0183, PN0939 and pipetted to mix. A compound wasprepared by: in a centrifuge tube, 172.00 of 10 mM Hepes/5% dextrosebuffer (pH 5.0) was added first, then 10 μl Inm4 (5 mg/ml, in 10 mMHepes/5% dextrose buffer of pH 5.0). Pipetted to mix. Later 11.20 μl ofPN0183 (5 mg/ml, in 10 mM Hepes/5% dextrose buffer of pH 5.0) was added.Pipetted to mix. Finally added 6.80 μl of PN0939 (5 mg/ml, in 10 mMHepes/5% dextrose buffer of pH 5.0). Pipetted again to mix. The solutionwas equilibrated on ice for 1 hr.

Preparation Example 15

Inm4, order of siRNA, PN0183, PN0939 and vortexed to mix. A compound wasprepared by: in a centrifuge tube, 2289.50 μl of 10 mM Hepes/5% dextrosebuffer (pH 5.0) was added first, then 24.00 μl of Inm4 (20 mg/ml, inRNase free water). Vortexed to mix. Later 53.60 μl of PN0183 (10 mg/ml,in 10 mM Hepes/5% dextrose buffer of pH 5.0) was added. Vortexed to mix.Finally 32.90 μl of PN0939 (20 mg/ml, in 10 mM Hepes/5% dextrose bufferof pH 5.0) was added. Pipetted to mix. The solution was equilibrated onice for 1 hr.

Preparation Example 16

Inm4, order of siRNA, PN0183, PN0939 and pH 7.4. A compound was preparedby: in a centrifuge tube, 376.19 μl of 10 mM Hepes/5% dextrose buffer(pH 7.4) was added first, then 5 μl Inm4 (20 mg/ml, in RNase freewater). Vortexed to mix. Later 15.39 μl PN0183 (7.26 mg/ml, in RNasefree water) was added. Vortexed to mix. Finally 3.42 μl of PN0939 wasadded. Pipetted to mix.

Preparation Example 17

G1498, PN0183 and tert-Butanol. A compound was prepared by: in acentrifuge tube, 72.93 μl of 10 mM Hepes/5% dextrose buffer (pH 5.0) wasadded first, then 4.17 μl PN0183 (5 mg/ml, in 10 mM Hepes/5% dextrosebuffer of pH 5.0). Vortexed to mix. Later added 10 μl of G1498 (1 mg/ml,in 10 mM Hepes/5% dextrose buffer of pH 5.0). Vortexed again to mix.Finally, added 12.90 μl of Tert-Butanol and pipetted to mix.

Preparation Example 18

G1498, PN0183 and ethanol. A compound was prepared: in a centrifugetube, 73.33 μl of 10 mM Hepes/5% dextrose buffer (pH 5.0) was addedfirst, then 4.17 μl PN0183 (5 mg/ml, in 10 mM Hepes/5% dextrose bufferof pH 5.0). Vortexed to mix. Later added 10 μl of G1498 (1 mg/ml, in 10mM Hepes/5% dextrose buffer of pH 5.0). Vortexed again to mix. Finally,added 12.50 μl of Ethanol and pipetted to mix.

Preparation Example 19

Lac-Z, PN0183, PN0939. A compound was prepared by: diluted 5.0 μl ofLac-Z siRNA (20 μM) into 120 μl OPTI-MEM medium. Added 1.62 μl PN0183 (1mg/ml) and 1.98 μl PN0939 (1 mg/ml) into 121.40 μl of OPTI-MEM medium.Combine the two solutions and pipetted to mix.

The structure of Lac-Z is:

Sense: CN2938. 5′-r(CUACACAAAUCAGCGAUUU)dTdT-3′ (SEQ ID NO: 64)Antisense: CN2939. 5′-r(AAAUCGCUGAUUUGUGUAG)dTdC-3′ (SEQ ID NO: 65)

Preparation Example 20

Lac-Z, PN0183, PN0938. A compound was prepared by: diluted 5.0 μl ofLac-Z siRNA (20 μM) into 120 μl OPTI-MEM medium. Added 1.62 μl PN0183 (1mg/ml) and 0.97 μl PN0938 (1 mg/ml) together into 122.41 μl of OPTI-MEMmedium. Combine the two solutions and pipetted to mix.

Preparation Example 21

Lac-Z, PN0183, PN0939 and crosslinking. A compound was prepared by:Diluted 5.0 μl of Lac-Z siRNA (20 μM) into 120 μl OPTI-MEM medium. Added1.62 μl PN0183 (1 mg/ml) and 1.98 μl PN0939 (1 mg/ml) into 119.80 μl ofOPTI-MEM medium. Combined the two solutions and pipetted to mix. Thenadded 1.60 μl of Glutaraldehyde (0.05%, W/V) and pipetted to mix. Thesolution was equilibrated at room temperature for 1 hr.

Preparation Example 22

Lac-Z, PN0183, crosslinking and PN0939. A compound was prepared: Diluted5.0 μl of Lac-Z siRNA (20 μM) into 120 μl OPTI-MEM medium. Added 1.62 μlPN0183 (1 mg/ml) into 119.80 μl of OPTI-MEM medium. Combined the twosolutions and then added 1.60 μl of Glutaraldehyde (0.05%, W/V).Pipetted to mix. This solution was equilibrated at room temperature for1 hr. Finally, added 1.98 μl PN0939 (1 mg/ml). Pipetted to mix.

Preparation Example 23

Lac-Z, PN0183, crosslinking, PN0939, and crosslinking. A compound wasprepared by: Diluted 5.0 μl of Lac-Z siRNA (20 μM) into 120 μl OPTI-MEMmedium. Added 1.62 μl PN0183 (1 mg/ml) into 119.80 μl of OPTI-MEMmedium. Combined the two solutions and then added 0.8 μl ofGlutaraldehyde (0.05%, W/V). Pipetted to mix. This solution wasequilibrated at room temperature for 1 hr. And then added 1.98 μl PN0939(1 mg/ml) and 0.8 μl of Glutaraldehyde (0.05%, W/V). Pipetted to mix.

Preparation Example 24

Lac-Z, PN0183, crosslinking, dialysis and PN0939. A compound wasprepared by: Made the Lac-Z siRNA and PN0183 combination first by adding158.6 μl of 10 mM Hepes/5% dextrose buffer (pH 7.4), 103.45 μl of Lac-ZsiRNA (20 μM) and 33.53 μl PN0183 (1 mg/ml). Vortexed to mix. Then added4.4 μl of Glutaraldehyde (0.05%, W/V). Pipetted to mix. This solutionwas equilibrated at room temperature for 2 hrs. Then the solution wasdialyzed at 4° for overnight. Diluted 43.5 μl of crosslinked combinationinto 331.5 μl of OPTI-MEM. Diluted 4.96 μl of PN0939 (0.1 mg/ml) into57.54 μl of OPTI-MEM. Combine the two diluted solutions and pipetted tomix.

Preparation Example 25

Lac-Z, PN0183, PN0826 and PEG 3350. A compound was prepared by: added5.0 μl of Lac-Z siRNA (20 μM) and 1.6 μl PN0183 (0.1 mg/ml) into 120 μlOPTI-MEM medium and vortexed to mix. Added 3.96 μl PN0826 (0.1 mg/ml)and 2.50 μl of PEG 3350 (10 mg/ml) into 118.54 μl of OPTI-MEM medium.Combine the two solutions and pipetted to mix.

Example 1 Gold Dye Displacement Assay for Peptide-siRNA Affinity

The relative binding of various peptides to siRNA via a rapid screen wasassessed by indirect measurement of the displacement of SYBR-goldnucleic acid binding dye. A buffered mixture of siRNA, peptide andSYBR-gold was prepared in the measurement plate in duplicate such thatthe peptide and SYBR-gold dye underwent simultaneous competitive bindingof the siRNA. The concentration of siRNA was fixed at 10 μg/mL and wascombined with a titration of each peptide ranging in a concentrationthat corresponded to a peptide:siRNA charge ratio between 0.05 and 10.Since SYBR-gold dye only fluoresces when bound to siRNA, peptide bindingto the siRNA inhibits binding of the dye and consequently reduces thefluorescence. Therefore, the amount of fluorescence correlated inverselyto the binding of the peptide to the siRNA. Both Kd and B_(max) valueswere calculated. A greater Kd value indicated greater binding affinitybetween the peptide and the siRNA.

SYBR-gold nucleic acid binding dye stock, a 10,000× concentrate, wassupplied by Invitrogen (Carlsbad, Calif.) and stored at −20° C. Theconcentrate was allowed to equilibrate to room temperature beforediluting 1 to 100 in Hyclone nuclease free water. This was diluted 1 to10 in the experimental plate for a final concentrate of 10× for theassay. This was the optimal dilution to achieve linear binding to siRNAduplex at a concentration range of up to 50 μg/mL concentration. Thevalues used to generate the standard curve demonstrating linear bindingof SYBR-gold to G1498 siRNA are shown in Table 4.

TABLE 4 G1498 siRNA Standard Curve Values [G1498] Mean μg/mLFluorescence Std Dev 0 0 3.86 1.56 376 10.0 3.13 840 44.8 6.25 3254 91.412.5 10591 762 25.0 26276 1497 50.0 36543 240

Samples were mixed directly in the 384 well analysis plate. First, 5 μLSYBR-gold dye was pipetted into each well with a multichannel pipet,touching the tip to the bottom of the well to draw out the solutioncompletely. Second, 22.5 μL, of 2× peptide solution was added with asingle channel pipet. Finally, 22.5 μL of 2× siRNA was added with amultichannel pipet. The plate was covered immediately with foil andtapped gently to mix and draw down any droplets on the side of the well.

Fluorescence was measured using the SpectraMax fluorescent plate readerfrom Molecular Devices (Sunnyvale, Calif.). Plate settings includedshaking before reading, one read per well, with excitation wavelength of495 nm and emission wavelength of 537 nm. The plate was read within 30minutes of the addition of the siRNA.

Scatchard Plot for Peptide Binding

A Scatchard Plot is a plot of peptide binding([peptide]bound/[peptide]free) vs. [peptide]bound. The slope of thelinear regression of this plot is −1/Kd and Bmax is the y-intercept.Since the concentration of free and bound peptide cannot be measureddirectly, indirect measurement of siRNA was used for the calculation.Free siRNA was determined from measured fluorescence using the standardcurve. Bound siRNA was determined from the standard curve by massbalance from the known initial siRNA concentration (10 μg/mL).

Bound peptide was calculated from bound siRNA by assuming the(siRNA:Peptide) bound molar ratio was equal to the (siRNA:Peptide)charge ratio for single molecule pairing. From this calculated boundpeptide amount, the free peptide was calculated by mass balance.

Particle Size and Zeta Potential

Particle size and zeta potential were determined with a MalvernZetasizer Nano ZS (Malvern, Worcestershire, UK) using a DTS1060C cleardisposable zeta cell at 25°. The dispersant for particle size was PBS,1.0200 CP viscosity, or water, 0.8872 CP viscosity. The dispersant forzeta potential was water 0.8872 CP viscosity. The dispersant viscositywas used as the sample viscosity. When both the zeta potential and theparticle size were measured, the clear disposable zeta cell was used.When only the particle size was measured, then a low volume disposablesizing cuvette was used.

Example 2 Condensate Particle Size at Various Nucleic AcidConcentrations and N/P Ratios

Diameters of particles of a condensate compound of siRNA G1498 andpeptide PN183 at various concentrations of G1498 and various N/P ratiosare shown in FIG. 1. For each group of three bars at a particular N/P,the concentration of G1498 for the leftmost bar was 100 ug/ml, for themiddle bar was 50 ug/ml, and for the rightmost bar was 10 ug/ml. At N/Pof 0.2 and 0.5, the particles were very small when the concentration ofG1498 was 10 ug/ml, thus the bar does not appear.

At N/P ratios below about 1.4, the particle size was below about 200 nmfor all concentrations of the siRNA. At N/P ratios at or above about1.4, condensate particle size remained below about 200 nm for allconcentrations of RNA except the highest (100 ug/ml).

Example 3 Condensate Particle Size at Various Nucleic AcidConcentrations and N/P Ratios

Diameters of particles of condensate compounds of siRNA G1498 andpeptide PN183 obtained at various times after mixing and at variousnitrogen to phosphorous ratios (N/P) are shown in FIGS. 2-5. In each ofFIGS. 2-5, for each group of two bars at a particular N/P ratio, theleft bar was with vortexing, while the right bar was without vortexing.

The particles sizes in FIG. 2 were obtained immediately after mixing,while those of FIGS. 3, 4, and 5 were obtained 30 minutes, 60 minutes,and 24 hours after mixing, respectively.

Example 4 Effect of pH on Condensate Particle Size

Diameters of particles of condensate compounds of siRNA G1498 andpeptide PN183 obtained at an N/P ratio of 1.4 and at a concentration ofG1498 of 100 ug/ml for various values of pH are shown in FIG. 6.

At pH below about 12, condensate particle size decreases, and continuesto decrease as pH decreases. Particle size was below about 500 nm for pHbelow about 11.

The intensity is the measure of back scattered photons (backscattermode). The particle size is calculated size using an algorithm for thediffusion autocorrelation.

Example 5 Effect of Salt Concentration on Condensate Particle Size

Diameters of particles of condensate compounds of siRNA G1498 andpeptide PN183 obtained at various concentrations of sodium chloride areshown in FIG. 7.

At sodium chloride concentrations up to about 0.5, particle sizeincreases from about 100 nm to about 275 nm. At sodium chlorideconcentrations greater than about 0.5, condensate particle sizefluctuates.

Example 6 Effect of Order of Addition of RNA and Peptide on CondensateParticle Size

Diameters of particles of condensate compounds of siRNA G1498 withpeptide PN183 at various N/P ratios and order of mixing are shown inFIG. 8. At N/P ratios at or below about 0.5, particle size was not muchaffected by the order of addition. At N/P ratios above about 0.5,particle size was generally smaller when the siRNA was introduced to thesolution first, and the peptide was added to the siRNA solution.

Example 7 Morphology of Condensate Particles

The morphology of the particles of a peptide-RNA condensate compound wasdetermined by transmission electron microscopy (TEM) imaging. Thefollowing protocol was used:

15 uL sample drop on top of grid, 10 min; Half strength Karnofsky'ssolution dip; Cacodylate buffer dip; TEM Contrast agent: 3% UranylAcetate (UA); Water dip, 3X, UA dip, blot off excess on damp filterpaper, dry. Mixture 1: (Original Karnovsky's Mixture); 16%Paraformaldehyde Solution: 20 mL; 50% Glutaraldehyde EM Grade:  8 mL;0.2M Sodium Phosphate Buffer: 25 mL; Distilled Water: 25 mL.

Final mixture is 78 mL with 5% Glutaraldehyde, 4% Formaldehyde in 0.08Mbuffer.

This mixture had an osmolarity of more than 2000 m OSM.

Sodium Cacodylate Buffers  0.1M; Sodium cacodylate: 4.28 gm; Calciumchloride: 25.0 gm; 0.2N hydrochloric acid:  2.5 ml; Dilute to 200 mlwith distilled water, pH 7.4.

Using the protocol above with no glow discharge, a TEM of particles of acondensate compound of siRNA G1498 (concentration 100 ug/ml) withpeptide PN183 (N/P=1.4) is shown in FIG. 9. This image shows particlesof uniform size and spherical morphology. The particle size was below100 nm, typically about 50-60 nm.

Using the protocol above with glow discharge, a TEM of particles of acondensate compound of siRNA G1498 (concentration 100 ug/ml) withpeptide PN183 (N/P=1.4) is shown in FIG. 10. This image shows particlesof uniform size and spherical morphology. The particle size is below 100nm, typically about 30-60 nm.

Example 8 Particle Size Characteristics of Peptide-RNA CondensateCompounds

The particle size characteristics of some peptide-RNA condensatecompounds are summarized in Table 5.

TABLE 5 Particle Size Characteristics of Peptide-RNA CondensatesParticle sizes at half Z-avg. Zeta peak height in nm diameter PotenialCompound N/P (% of population) (nm) (mV) G1498/PN183   0.5 40-106(98.7%)  63.8 −35.7   (100%) G1498/PN183 2 50-110 (100%)  93.2 27.9(100%) G1498/PN826 2 103-120 (92.7%)  189 peak 34.6 G1498/PN183/PN8260.5; 1 90-145 (47.4%)  119 peak 31.3 190-340 (52.6%)  268 peak (100%)G1498/PN861 2 180-330 (98.7%)  241 — G1498/PN939 2 20-70 (92.1%)  37.0 —G1498/PN938 2 <10 (32.3%) <10 — 180-500 (56.8%)  283 peakG1498/PN183/PN939 0.5; 1 <1 (9.8%)  0.8 peak — 15-35 (68.5%) 23.2 peak200-400 (21.7%)  274 peak G1498/PN924 2 <1 (41.4%)  0.8 peak — 5-8(11.3%)  6.2 peak 80-200 (47.3%)  135 peak G1498/PN859 2 530-770 (94.7%) 702 —

For example, compound G1498/PN183 at N/P of 0.5 exhibited a peak having98.7% of total intensity, 73.3 nm peak diameter, 32.9 mm peak width, andZ-average diameter 63.8.

Example 9 In vivo Knockdown Assay of TFN-α in LPS-Stimulated Mouse LungUsing Peptide-RNA Condensates

SiRNA knockdown activity was determined by transfecting cells with apeptide-siRNA condensate compound. A random siRNA sequence was used as anegative control.

FIG. 11 shows the results of a knockdown assay of LPS-induced TFN-αexpression (pg/ml) in a mouse model by intranasal administration of acomposition including a condensate compound of siRNA Inm-4 and peptidesPN183 and PN939.

In FIG. 11, buffer control is the leftmost bar, followed by data forcondensate Inm-4/PN183/PN939, and followed on the right by data forcompound Inm-4/PN183/PN939 crosslinked with glutaraldehyde (G). Placebodoes not contain the siRNA, and Qneg contains a non-active-siRNA.

The data for FIG. 11 are given in Table 6.

TABLE 6 Knockdown of TFN-α in LPS-Stimulated Mouse Lung With Inm-4 siRNALPS Lung Assay Compound Assay Mean SD Buffer (10 mM Hepes/5% Dextrose)211.3 25 PN183/PN939 Placebo 188.3 10 N/P = 0.75/0.5 Qneg 207.1 95 Inm-4agent 97.0 55 PN183/PN939/G Placebo 179.1 51 N/P = 0.75/0.5 Qneg 161.7108 G = 0.8 ME Inm-4 agent 119.4 6

Dose was administered intranasally. At 4 and 24 hrs post dose, animalswere induced with 0.625 ng LPS (50 μl). Lungs were collected 2 hrs.post-LPS. A TNFα ELISA assay and a BCA total protein assay wereperformed. Materials and methods for the assay were as follows:

-   -   Animals: Normal mice    -   Dose: 0.5 mg/kg    -   Volume: 50 uL    -   Replicates: n=3    -   Total Groups: 10    -   Controls: Vehicles, Qneg    -   siRNA: Inm4    -   Dosing: Formulations were prepared at 0.5 mg/kg with Inm4. Each        formulation had 200 ul in total. Each mouse (n=3) received 50        ul.    -   siRNA preparation: Existing 20 mg/ml stock of Inm-4 siRNA was        diluted into 5 mg/ml using Hepes/Buffer. Existing 3.29 mg/ml        stock of Qneg was used.    -   Peptides: Peptides were diluted to appropriate concentration        using Buffer (10 mM Hepes/5% dextrose).    -   Excipient Preparation Glutaraldehyde (0.05% W/V) was used, 0.2        um filtered to be sterile.    -   Formulation Preparation: Components were added to Bio-pur        Eppendorf 1.5 ml tubes.    -   (A) Buffer was added first, as a receiving volume for small        volumes of other components.    -   (B) All the components were added in the following order: siRNA,        peptide-1, peptide-2, additive, if any, buffer.    -   (C) For formulations with glutaraldehyde crosslinking, waited        for one hour before dosing.

Representative formulations are given in Table 7.

TABLE 7 Formulations for Peptide-siRNA Compounds Vol. of Vol. of Vol. ofsiRNA peptide peptide Vol. of Vol. of stock stock-1 stock-2 additiveBuffer Total Vol. Formulation (ul) (ul) (ul) (ul) (ul) (ul) Buffer (10mM Hepes, 5% 0 0 0 0 200 200 Dextrose) PN0183/PN0939 0.00 11.20 6.800.00 182.00 200 N/P = 0.75, N/P = 0.5 PN0183/PN0939/G 0.00 11.20 6.8034.48 147.52 200 N/P = 0.75, N/P = 0.5, 0.8 mole equivalentQ.Neg/PN0183/PN0939 15.20 11.20 6.80 0.00 166.80 200 N/P = 0.75, N/P =0.5 Q.Neg/PN0183/PN0939/G 15.20 11.20 6.80 34.48 132.32 200 N/P = 0.75,N/P = 0.5, 0.8 mole equivalent Inm4/PN0183/PN0939 10.00 11.20 6.80 0.00172.00 200 N/P = 0.75, N/P = 0.5 Inm4/PN0183/PN0939/G 10.00 11.20 6.8034.48 137.52 200 N/P = 0.75, N/P = 0.5, 0.8 mole equivalent

Details of the representative formulations are given in Tables 8 and 9.

TABLE 8 siRNA final concentration = 250 ug/ml (0.5 mg/kg) siRNA Inm4stock conc. = 5 mg/ml Qneg. = 3.29 mg/ml PN0183 = 5 mg/ml PN0939 = 5mg/ml Glutaraldehyde = 0.05% W/V

TABLE 9 Materials Batch ID Concentration Inm4 BS31 20 mg/ml Q.NegB324P167 3.29 mg/ml PN0183-2 BP1 10 mg/ml PN0939-2 BP9 20 mg/mlGlutaraldehyde BR39 0.05% W/V Buffer BB72 10 mM Hepes/5% Dextrose

Example 10 In vitro Knockdown Assay of Lac-z Expression in RatGliosarcoma Fibroblast Cells (9L/LacZ)

FIG. 12 shows the results of a knockdown in vitro assay of lac-zexpression in rat gliosarcoma fibroblast cells 9L/LacZ for condensatecompounds of the lac-z siRNA with peptide PN183 and various secondpeptides.

In FIG. 12, comparative data using HiPerFect™ (Qiagen; Valencia, Calif.)is the leftmost bar, followed by data for various compounds of thisinvention. The N/P ratio for PN183 was 0.75, while the N/P ratio for thesecond peptide was 0.3. The data for FIG. 12 are given in Table 10.

TABLE 10 Lac-z Knockdown in vitro Assay Knockdown mean SD HiPerFect0.221048 0.028369 PN0183 (N/P = 0.75)/peptide PN0939 0.905998 0.053035 2(N/P = 0.3) PN0938 1.007354 0.1546 PN0826 0.762651 0.069725 PN09510.629382 0.128045 PN0970 0.806908 0.11293 PN0526 0.682695 0.045614

Materials and methods for the assay were as follows:

-   -   Cells: 9L/LacZ    -   Dose: 100 nM; based on 100 ul total transfection volume.    -   Volume: 25 uL formulation volume.    -   Replicates: n=3.    -   Total Groups: 20.    -   Controls: Qneg w/ Alexis 546.    -   siRNA: LacZ.    -   Lac-Z or Qneg: 54 ul siRNA+17.28 ul PN0183+1278 ul OPTI-MEM.    -   Peptides were diluted into appropriate concentration using        OPTI-MEM medium. All excipients were 0.2 um filtered to be        sterile.    -   Formulation:    -   (A) Diluted siRNA and PN0183 together using OPTI-MEM to form        particles. Vortexed.    -   (B) Diluted delivery vehicles using OPTI-MEM. Vortexed to mix        the delivery vehicle.    -   (C) For each formulation, in 96 well, added diluted delivery        vehicle first and then added siRNA/PN0183 formulation. Pipetted        to mix. Waited for 30 mins. before sending for transfection.        Transfection: Each formulation was 125 ul which was enough for 5        wells. Each well (n=3) received 25 ul.

Representative formulations are given in Table 11.

TABLE 11 Total vol Vol of for Vol of Vol of Optimem delivery peptidepeptide Vol of for additive substances Formul. stock1 for stock2 foradditives dilution dilution with 6 wells 10 wells for 10 (10 well; (10well; PN183 (ul) (ul) wells (ul) ul) ul) PN939 0.96 5.95 0.00 119.05125.00 PN938 0.96 2.91 0.00 122.09 125.00 PN826 0.96 3.96 0.00 121.04125.00 PN951 0.96 3.12 0.00 121.88 125.00 PN970 0.96 25.85 0.00 99.15125.00 PN526 0.96 6.90 0.00 118.10 125.00

Details of the representative formulations are given in Table 12.

TABLE 12 Materials Batch ID Concentration Lac-Z Qiagen (Cat 1027020; Lot161545/161546); 20 uM Q.Neg (Alexis 546) Qiagen (Cat 1027098; Lot160427/160428); 20 uM PN0183-2 BP86 7.26 mg/ml PN0939-2 BP9   20 mg/mlPN0826-2 BP2   5 mg/ml PN0951-2 BP10   10 mg/ml PN0970-1 462-124   2mg/ml PN0526-2 3.91 mg/ml Buffer BB72 10 mM Hepes/5% Dextrose

Example 11 In vitro Knockdown Assay of Lac-z Expression in RatGliosarcoma Fibroblast Cells (9L/LacZ)

Table 13 shows the results of knockdown in vitro assays of lac-zexpression in rat gliosarcoma fibroblast cells 9L/LacZ for variouscondensate compounds.

TABLE 13 Knockdown of Lac-z Expression in Rat Model Cell Line 9L/LacZRelative protein concentration N/P LacZ assay (Qneg) Compound ratiosMean SD Mean SD HiPerFect ™ — 0.165 0.028 0.413 0.057 PN0183/G(0.25 mol.eq., 0.75/2   0.510 0.071 1.059 0.121 dialyzed)/PN0951 PN0183/G(0.25mol. eq., 0.75/5   0.613 0.194 1.051 0.150 dialyzed)/PN0951PN0183/G(0.25 mol. eq., 0.75/0.5 0.725 0.129 1.146 0.183dialyzed)/PN0939 PN0183/G(5 mol. eq., 0.75/0.5 0.524 0.107 1.218 0.042dialyzed)/PN0939

Materials and methods for the assay were as follows:

-   -   Cells: 9L/LacZ.    -   Dose: 100 nM; based on 100 ul total transfection volume.    -   Volume: 25 uL formulation volume.    -   Replicates: n=3.    -   Total Groups: 20.    -   Controls: Qneg w/ Alexis 546.    -   siRNA: LacZ.    -   Transfection: Each formulation had 125 ul which was enough for 5        wells. Each well (n=3) received 25 ul.    -   Peptide Preparation Peptide was diluted into appropriate        concentration using OPTI-MEM medium.    -   Excipient Preparation All the excipients were 0.2 um filtered to        be sterile.    -   Formulation Preparation:    -   (A) For formulations without PN0183, added delivery vehicle        first and then siRNA, pipetted to mix.    -   (B) For formulations with PN0183, made siRNA and PN0183 complex        first. In 96 well plate, added delivery vehicles first, and then        siRNA/PN0183 complex, pipetted to mix.    -   (C) For formulations with crosslinking, make the siRNA/PN0183        complex first, then either dialysis (at 4°, overnight), or        without dialysis. Then next morning, added other delivery        vehicles first and siRNA/PN0183 complex, and then pipetted to        mix.

Representative formulations are given in Table 14.

TABLE 14 Vol of Volume of Optimem for Total vol for delivery deliverydelivery reagents for reagents substances 10 wells dilution (10 dilution(10 Code Formulation (ul) well; ul) well; ul) U PN0183/G/PN0951 20.82104.18 125.00 N/P = 0.75, 0.25 ME, D, N/P = 2 V PN0183/G/PN0951 52.0572.95 125.00 N/P = 0.75, 0.25 ME, D, N/P = 5 W PN0183/G/PN0939 9.92115.08 125.00 N/P = 0.75, 0.25ME, D, N/P = 0.5 Y PN0183/G/PN0939 9.92115.08 125.00 N/P = 0.75, 5ME, D, N/P = 0.5

Details of the representative formulations are given in Tables 15, 16,and 17.

TABLE 15 Crosslinking: Prepared siRNA/PN0183 complex first. 0.25MEcrosslinking: 103.45 ul of stock siRNA (20 uM) + 33.53 ul PN0183 (1mg/ml) + 4.4 Glutar. (0505%) + 158.6 ul Hepes Buffer. 5 ME crosslinking:68.97 ul siRNA stock (20 uM) + 22.35 ul PN0183 (1 mg/ml) + 5.9 ul ofGlutar. (0.5%) + 102.78 ul Hepes Buffer. 5 ME without dialysis: 17.24 ulsiRNA (20 uM) + 5.59 ul PN0183 (1 mg/ml) + 1.48 ul of Glutar. (0.5%) +25.69 ul Hepes Buffer. For 0.25 ME crosslinking: 43.5 ul crosslinkedcomplex + 331.5 OPTI-MEM. For 5ME crosslinking: 8.7 ul crosslinkedcomplex + 66.3 ul OPTI-MEM.

TABLE 16 G = Glutaraldehyde ME = Molar Equivalent D = DialysisCrosslinking = 2 hrs at room temperature PN0951 = 0.1 mg/ml PN0939 = 0.1mg/ml OPTI-MEM

TABLE 17 Materials Batch ID Concentration Lac-Z Qiagen (Cat 1027020; Lot161545/161546); 20 uM Q.Neg (Alexis 546) Qiagen (Cat 1027098; Lot160427/160428); 20 uM PN0951-2 BP10   10 mg/ml PN0183-2 BP86 7.26 mg/mlPN0939 BP9   20 mg/ml Buffer 10 mM Hepes/5% Dextrose, pH 7.4 OPTI-MEMBB57

Example 12 Polynucleotide Delivery-Enhancing Polypeptides

The exemplary polynucleotide delivery-enhancing polypeptide PN73 wasderived from the amino acid sequence of the human histone 2B (H2B)protein, which is shown below. The underlined residues 13 through 48found within H2B protein identify the fragment used to derive PN73. Itmay also be represented by H2B amino acids 12 through 48. The primarystructure of PN73 is also shown below.

H2B (histone 2B) amino acid sequence (SEQ ID NO: 66)MPEPAKSAPAPKKGSKKAVTKAQKKDSKKRKRSRKESYSVYVYKVLKVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPGELAKHAVSEGTKAVTKYTSSK PN73 (13-48) (SEQ ID NO: 42)NH2-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide

Table 18 shows the structure of some mutant polynucleotidedelivery-enhancing polypeptides made by residue substitutions anddeletions of the exemplary polynucleotide delivery-enhancing polypeptidePN73.

TABLE 18 PN73 Residue Substitution and Deletion Series SEQ ID PeptideNO: Amino Acid Sequence PN73 42 KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQPN644 67 KGSKKAVTKAQKKDGKKRKRSRKESYWVYVYKVLKQ PN645 68KGSKKAVTKAQKKDGKKRKRSRKWSYSVYVYKVLKQ PN646 69KGSKKAVTKAQKKDGKKRKRSRKFSYSVYVYKVLKQ PN647 70KGSFKAVTKAQKKDGKKRKRSFKFSYSVYVYKVLKQ PN729 71KGSFKAVTKAQKKFGKKRKRSRKSFSVYVYKVLKQ

Table 19 shows the structure of exemplary polynucleotidedelivery-enhancing polypeptide PN73 and truncated derivatives thereof.The amino acids sequence for PN360 and PN361 listed below are alignedwith the corresponding amino acid sequence of PN73.

TABLE 19 PN73 Deletion Series SEQ C-Term. ID Label Peptide NO: AminoAcid Sequence None PN73 42 KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amidePN360 72 KGSKKAVTKAQKKDGKKRKRSRK-amide PN361 73KKDGKKRKRSRKESYSVYVYKVLKQ-amide PN766 74 RKESYSVYVYKVLKQ-amide (PN708)FITC (fluorescein-5- PN690 75KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-GK[EPSILON-5CFG- isothiocyanate)label (PN73) amide (i.e., - GK[EPSILON]G- PN661 76KKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-GK[EPSILON-5CFG-amide amide) PN685 77VTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-GK[EPSILON-5CFG amide PN660 78AQKKDGKKRKRSRKESYSVYVYKVLKQ-GK[EPSILON-5CFG-amide PN735 79KDGKKRKRSRKESYSVYVYKVLKQ-GK[EPSILON-5CFG-amide PN655 80KKRKRSRKESYSVYVYKVLKQ-GK[EPSILON-5CFG-amide PN654 81KRSRKESYSVYVYKVLKQ-GK[EPSILON-5CFG-amide PN708 82RKESYSVYVYKVLKQ-GK[EPSILON-5CFG-amide PN653 83SYSVYVYKVLKQ-GK[EPSILON-5CFG-amide PN652 84VYVYKVLKQ-GK[EPSILON-5CFG-amide PN651 85 YKVLKQ-GK[EPSILON-5CFG-amidePN768 86 KVLKQ-GK[EPSILON-5CFG-amide

PN360 shares its N-terminus with PN73 but lacks PN73's C-terminus whilePN361 shares its C-terminus with PN73 but lacks PN73's N-terminus. PN766represents the 15 C-terminal amino acids of PN73. PN73, PN360, PN361 andPN766 are not tagged with a C-terminal FITC(fluorescein-5-isothiocyanate) (i.e., -GK[EPSILON]G-amide). Table 19further shows the 11 truncated forms of PN73 that were created bysequentially deleting 3 residues at a time, except PN768, from theN-terminus of the peptide. All these peptides were tagged with aC-terminus FITC (fluorescein-5-isothiocyanate) label (i.e.,-GK[EPSILON]G-amide) so that cells containing the peptide could bedetected by fluorescent microscopy and/or sorted by flow cytometry.PN766 and PN708 have the same amino acid sequence but differ in thatPN708 has the C-terminus FITC tag.

Example 13 In Vitro Methods and Procedures for siRNA Cell-Uptake andTarget Gene Knockdown

The present example illustrates the methods and procedures used toassess the efficacy of the exemplary polynucleotide delivery-enhancingpolypeptides listed in Table 18 and Table 19 of Example 12 to enhancesiRNA cell-uptake and siRNA mediated target gene knockdown activities.Cell viability was also assessed. The cell culture conditions andprotocols for each assay are explained below in detail.

Cell Cultures

Primary Human Monocytes: Fresh human blood samples from healthy donorswere purchased from Golden West Biologicals. For isolation of monocytes,blood samples were diluted with PBS at a 1:1 ratio immediately afterreceiving. Peripheral blood mononuclear cells (PBMC) were first isolatedby Ficoll (Amersham) gradient from whole blood. Monocytes were furtherpurified from PBMCs using the Miltenyi CD14 positive selection kit andsupplied protocol (MILTENYI BIOTEC). To asses the purity of the monocytepreparation, cells were incubated with an anti-CD14 antibody (BDBiosciences) and then sorted by flow cytometry. The purity of themonocyte preparation was greater than 95%.

Activation of human monocytes was performed by adding 0.1-1.0 ng/ml ofLiposaccharides, LPS (Sigma, St Louis, Mo.) to the cell culture tostimulate tumor necrosis factor-±(TNF-±) production. Cells wereharvested 3 hours after incubation with LPS and mRNA levels weredetermined by Quantigene assay (Genospectra, Fremont, Calif.) accordingto the manufacturer's instructions.

Mouse Tail Fibroblast Cells: Mouse tail fibroblast (MTF) cells werederived from the tails of C57BL/6J mice. Tails were removed, immersed in70% ethanol and then cut into small sections with a razor blade. Thesections were washed three times with PBS and then incubated in a shakerat 37° C. with 0.5 mg/mL collagenase, 100 units/mL penicillin and 100μg/mL streptomycin to disrupt tissue. Tail sections were then culturedin complete media (Dulbecco's Modified Essential Medium with 20% FBS, 1mM sodium pyruvate, nonessential amino acids and 100 units/mL penicillinand 100 μg/mL streptomycin) until cells were established. Cells werecultured at 37° C., 5% CO₂ in complete media as outlined above.

Cell Viability (MTT Assay)

Cell viability was assessed using the MTT assay (MTT-100, MatTek kit).This kit measures the uptake and transformation of tetrazolium salt toformazan dye. Thawed and diluted MTT concentrate was prepared 1 hourprior to the end of the dosing period with the lipid by mixing 2 mL ofMTT concentrate with 8 mL of MTT diluent. Each cell culture insert waswashed twice with PBS containing Ca⁺² and Mg⁺² and then transferred to anew 96-well transport plate containing 100 μL of the mixed MTT solutionper well. This 96-well transport plate was then incubated for 3 hours at37° C. and 5% CO₂. After the 3 hour incubation, the MTT solution wasremoved and the cultures transferred to a second 96-well feeder traycontaining 250 μL MTT extractant solution per well. An additional 150 μLof MTT extractant solution was added to the surface of each culture welland the samples sat at room temperature in the dark for a minimum of 2hours and maximum of 24 hours. The insert membrane was then pierced witha pipet tip and the solutions in the upper and lower wells were allowedto mix. Two hundred microliters of the mixed extracted solution alongwith extracted blanks (negative control) was transferred to a 96-wellplate for measurement with a microplate reader. The optical density (OD)of the samples was measured at 570 nm with the background subtraction at650 nm on a plate reader. Cell viability was expressed as a percentageand calculated by dividing the OD readings for treated inserts by the ODreadings for the PBS treated inserts and multiplying by 100. For thepurposes of this assay, it was assumed that PBS had no effect on cellviability and therefore represented 100% cell viability.

siRNA Preparation

Synthesis of oligonucleotides was carried out using the standard2-cyanoethyl phosphoramidite method (1) on long chain alkylaminecontrolled pore glass derivatized with5′-O-Dimethyltrityl-2′-O-t-butyldimethylsilyl-3′-O-succinylribonucleoside of choice or 5′-O-Dimethyltrityl-2′-deoxy-3′-O-succinylthymidine support where applicable. All oligonucleotides weresynthesized at either the 0.2 or 1-μmol scale using an ABI 3400 DNA/RNAsynthesizer (Applied Biosystems, Foster City, Calif.), cleaved from thesolid support using concentrated NH₄OH, and deprotected using a 3:1mixture of NH₄OH:ethanol at 55° C. The deprotection of 2′-TBDMSprotecting groups was achieved by incubating the base-deprotected RNAwith a solution (600 μL per μmol) ofN-methylpyrrolidinone/triethylamine/triethylamine trihydrofluoride(NMP/TEA/3HF; 6:3:4 by volume) at 65° C. for 2.5 hours. Thecorresponding building blocks,5′-dimethoxytrityl-N-(tac)-2′-O-(t-butyldimethylsilyl)-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramiditesof A, U, C and G (Proligo, Boulder, Colo.) as well the modifiedphosphoramidites, 5′-DMTr-5-methyl-U-TOM-CE-Phosphoramidite,5′-DMTr-2′-OMe-Ac-C-CE Phosphoramidite, 5′-DMTr-2′-OMe-G-CEPhosphoramidite, 5′-DMTr-2′-OMe-U-CE Phosphoramidite,5′-DMTr-2′-OMe-A-CE Phosphoramidite (Glen Research, Sterling Va.) werepurchased directly from suppliers. Triethylamine-trihydrofluride,N-methylpyrrolidinone and concentrated ammonium hydroxide was purchasedfrom Aldrich (Milwaukee, Wis.). All HPLC analysis and purifications wereperformed on a Waters 2690 HPLC system with Xterra™ C18 columns. Allother reagents were purchased from Glen Research Inc. Oligonucleotideswere purified to greater than 97% purity as determined by RP-HPLC.siRNAs for mouse injection were purchased from Qiagen (Valencia, Calif.)as in-vivo grade, which were HPLC purified after annealing. The amountof single stranded siRNA was determined spectrophotometrically based ona calculated extinction coefficient of 35.0 μg/OD for the sodium saltform at λ=260 nm. When the two strands are annealed, approximately 10%hypochromicity was observed; therefore the extinction coefficient waslowered by 10% for the quantitation of the double stranded forms.Endotoxin levels of siRNAs were typically equal to or less than 0.0024EU/mg.

Peptide Synthesis

Peptides were synthesized by solid-phase Fmoc chemistry on CLEAR-amideresin using a Rainin Symphony synthesizer. Coupling steps were performedusing 5 equivalents of HCTU and Fmoc amino acid with an excess ofN-methylmorpholine for 40 minutes. Fmoc removal was accomplished bytreating the peptide resin with 20% piperidine in DMF for two 10 minutescycles. Upon completion of the entire peptide, the Fmoc group wasremoved with piperidine and washed extensively with DMF. Maleimidomodified peptides were prepared by coupling 3.0 equivalents of3-maleimidopropionic acid and HCTU in the presence of 6 equivalents ofN-methylmorpholine to the N-terminus of the peptide resin. The extent ofcoupling was monitored by the Kaiser test. The peptides were cleavedfrom the resin by the addition of 10 mL of TFA containing 2.5% water and2.5 triisopropyl silane followed by gentle agitation at room temperaturefor 2 h. The resulting crude peptide was collected by trituration withether followed by filtration. The crude product was dissolved inMillipore water and lyophilized to dryness. The crude peptide was takenup in 15 mL of water containing 0.05% TFA and 3 mL acetic acid andloaded onto a Zorbax Rx-C8 reversed-phase (22 mm ID×250 mm, 5 μmparticle size) through a 5 mL injection loop at a flow rate of 5 mL/min.The purification was accomplished by running a linear AB gradient of0.1% B/min where solvent A is 0.05% TFA in water and solvent B is 0.05%TFA in acetonitrile. The purified peptides were analyzed by HPLC andESMS.

Flow Cytometry

Fluorescence activated cell sorting (FACS) analysis were performed usingBeckman Coulter FC500 cell analyzer (Fullerton, Calif.). The instrumentwas adjusted according to the fluorescence probes used (FAM or Cy5 forsiRNA and FITC and PE for CD14). Propidium iodide (Fluka, St Louis) andAnnexinV (R&D systems, Minneapolis) were used as indicators for cellviability and cytotoxicity. A brief step-by-step protocol is detailedbelow.

-   -   (a) After exposure to the complex of siRNA/peptide, cells were        incubated for at least 3 hours.    -   (b) Wash cells with 200 μl PBS.    -   (c) Detach cells with 15 μl TE, incubate at 370C.    -   (d) Re-suspend cells in five wells with 30 μl FACS solution (PBS        with 0.5% BSA, and 0.1% sodium Azide).    -   (e) Combine all five wells into a tube.    -   (f) Add PI (Propidium iodide) 5 μl into each tube.    -   (g) Analyze the cells with fluorescence activated cell sorting        (FCAS) according to manufacturer's instructions.

For siRNA uptake analysis, cells were washed with PBS, treated withtrypsin (attached cells only), and then analyzed by flow cytometry.Uptake of the siRNA designated BA, described above, was also measured byintensity of Cy5 or FITC fluorescence in the cells and cellularviability assessed by addition of propidium iodide or AnnexinV-PE. Inorder to differentiate the cellular uptake from the membrane insertionof fluorescence labeled siRNA, trypan blue was used to quench thefluorescence on the cell membrane surface.

Example 14 Deletion Analysis of the Exemplary PolynucleotideDelivery-Enhancing Polypeptide

The efficacy of the full-length and truncated forms of polypeptide PN73to enter cells was tested in vitro by a cell-uptake assay with primarymouse tail fibroblast (MTF) cells. The number of cells in culture thatreceive the FITC-labeled peptide was measured by flow cytometry. Thepercentage peptide cell-uptake was expressed relative to the totalnumber of cells present in the culture. In addition, the MeanFluorescence Intensity (MFI) was used to evaluate the quantity ofFITC-labeled peptide found within cells. MFI directly correlates withthe amount of FITC-labeled peptide within the cell: higher relative MFIvalue correlates with a greater amount of intracellular FITC-labeledpeptides. Peptides were evaluated at 0.63 μM, 2.5 μM and 10 μMconcentrations; PN768 was tested at 2 μM, 10 μM and 50 μM.

Full-length and truncated forms of the exemplary polynucleotidedelivery-enhancing polypeptide PN73, were exposed to cells the daybefore transfection. FITC-tagged peptides were diluted in Opti-MEM®media (Invitrogen) for about 5 minutes at room temperature and thenadded to cells. Cells were transfected for 3 hours at and washed withPBS, treated with trypsin, and then analyzed by flow cytometry. Cellviability was determined as above. Cellular uptake was distinguishedfrom the membrane insertion using trypan blue to quench any fluorescenceon the cell membrane surface.

For the cell-uptake assay, the full-length FITC-labeled PN73 peptide(PN690) achieved nearly 100% cell uptake at all tested concentrations(10 μM results shown in Table 20 column entitled “% PeptideCell-Uptake”). The remaining truncated forms of PN73, at 10 μMconcentration except for PN768 which required 50 μM, achieved a percentcell uptake (values in parentheses) comparable to that of PN690indicating that the N-terminal residues of PN73 are not required for thepeptide's ability to enter cells. The five C-terminal residues of PN73,identified as PN768, are sufficient for peptide cell-uptake. Thetruncated forms of PN73 at 0.63 μM showed a decrease in cell uptakeactivity proportionate to the length of the peptide. In other words, thegeneral observation of the peptides tested at a 0.63 μM concentration isthat, as the PN73 peptide's length decreased, its cell uptake activitydecreased thus indicating peptide cell-uptake activity is dosedependent.

Table 20 summarizes data for cell uptake and target gene knockdown (KD).

TABLE 20 Summary of Functional Domain Analysis of the PN73 PeptideDeletion Series C- Term. % Peptide Peptide % siRNA siRNA Label PeptideCell-Uptake FITC MFI Cell-Uptake Cy5 MFI KD None PN73 N/A N/A 98% (10μM) 13 (10 μM) + PN360 N/A N/A 0% NT NT PN361 N/A N/A 55% (20 μM) NT NTFITC PN690 100 (10 μM) 125 (10 μM) 58 (2.5 μM) 50 (10 μM) +(fluorescein-5- (PN73) isothiocyanate) label PN661 100 (10 μM) 128 (10μM) 49 (2.5 μM) 59 (10 μM) NT (i.e., -GK[EPSILON]G- PN685 100 (2.5 μM)151 (10 μM) 24 (2.5 μM) 61 (10 μM) NT amide) PN660 100 (10 μM) 121 (10μM) 41 (2.5 μM) 68 (10 μM) + PN735 100 (10 μM) 82 (10 μM) 13 (2.5 μM) 38(10 μM) − PN655 100 (10 μM) 63 (10 μM) 14 (10 μM) 44 (10 μM) NT PN654 95(10 μM) 10 (10 μM) 27 (10 μM) 14 (10 μM) ± PN708 97 (10 μM) 10 (10 μM)42 (10 μM) 34 (10 μM) + PN653 95 (10 μM) 8 (10 μM) 1.7 (10 μM) 4 (10 μM)− PN652 86 (10 μM) 5 (10 μM) 1.8 (10 μM) 5 (10 μM) NT PN651 90 (10 μM) 5(10 μM) 0 3 (.65 μM) NT PN768 91 (50 □M) 9 (50 μM) NT NT NT NT = nottested; peptide concentrations (in parenthesis) given are those thatachieved the given uptake, in percent, or MFI in relative values.

Table 20 shows that deleting part of the N-terminus of PN73 (see PN361)reduced siRNA cell-uptake activity by 50%; and removal of C-terminalresidues (see PN360) reduced siRNA cell-uptake activity. These data showthat the C-terminal domain of the exemplary polynucleotidedeliver-enhancing polypeptide PN73 contributes to nucleotide cell-uptakeactivity of the peptide.

The effective knockdown of target gene expression bysiRNA/polynucleotide delivery-enhancing polypeptide complexes of theinvention was demonstrated. Specifically, the ability of siRNA/peptidecomplexes to modulate expression of the human tumor necrosis factor-α(hTNF-α) gene was assessed. The significance of targeting the hTNF-αgene is that it is implicated in mediating the occurrence or progressionof rheumatoid arthritis (RA) when over-expressed in human and othermammalian subjects.

Human monocytes were used as a model system to determine the effect ofsiRNA/peptide complexes on hTNF-α gene expression. Qneg represents arandom siRNA sequence and functioned as the negative control. Theobserved Qneg knockdown activity is normalized to 100% (100% geneexpression levels) and the knockdown activity of each of the followingsiRNAs A19S21, 21/21 and LC20 was presented as a relative percentage ofthe negative control. A19S21, 21/21 and LC20 are siRNAs that targethTNF-α mRNA. The exemplary polynucleotide delivery-enhancingpolypeptides PN643 (full-length PN73 minus a C-terminal label), PN690(full-length PN73 with a C-terminal FITC-label) and the truncated formsof PN73 from the deletion series, PN660, PN735, PN654 and PN708 werecomplexed with the above listed siRNAs to determine their effect on eachsiRNA's ability to reduce hTNF-α gene expression levels in humanmonocytes. The knockdown activity for the full length and truncatedforms of the exemplary polynucleotide delivery-enhancing polypeptidePN73 are summarized above in Table 20. A “+” in the “KD” columnindicates that the peptide/siRNA complex had knockdown activity of 80%of the Qneg negative control siRNA (20% reduction in mRNA levelscompared to the Qneg negative control). A “+/−” indicates that thepeptide/siRNA complex had a knockdown activity of approximately 90% ofthe Qneg negative control siRNA (10% reduction in mRNA levels comparedto the Qneg negative control). Finally, a “−” indicates that thepeptide/siRNA complex had no significant knockdown activity compared tothe Qneg negative control.

Healthy human blood was purchased from Golden West Biologicals (CA), theperipheral blood mononuclear cells (PBMC) were purified from the bloodusing Ficoll-Pague plus (Amersham) gradient. Human monocytes were thenpurified from the PBMCs fraction using magnetic microbeads from MiltenyiBiotech. Isolated human monocytes were resuspended in IMDM supplementedwith 4 mM glutamine, 10% FBS, 1× non-essential amino acid and 1×pen-strep, and stored at 4C until use.

In a 96 well flat bottom plate, human monocytes were seeded at100K/well/100 μl in OptiMEM medium (Invitrogen). Exemplarypolynucleotide delivery-enhancing polypeptides were mixed with 20 nMsiRNA at a molar ratio of 1 to 5 in OptiMEM medium at room temperaturefor 5 minutes. At the end of incubation, FBS was added to the mixture(final 3%), and 50 μl of the mixture was added to the cells. The cellswere incubated at 37° C. for 3 hours. After incubation, the cells weretransferred to V-bottom plate and pelleted at 1500 rpm for 5 min. Thecells were resuspended in growth medium (IMDM with glutamine,non-essential amino acid, and pen-strep). After an overnight incubation,the monocytes were stimulated by application of LPS (Sigma) at 1 ng/mlfor 3 hours to increase expression of TNF-α expression levels. Afterinduction by LPS, cells were collected as above for mRNA quantification,and supernatant was saved for protein quantification if desired.

For mRNA measurement, branch DNA technology from Genospectra (CA) wasused according to manufacturer's specification. To quantitate mRNA levelin the cells, both house keeping gene (cypB) and target gene (TNF-α)mRNA were measured, and the reading for TNF-α was normalized with cypBto obtain relative luminescence unit.

In general, PN643 (full-length non-FITC-labeled PN73) and PN690(full-length FITC-labeled PN73) had equivalent siRNA knockdownactivities for all siRNAs tested as indicated by “+” in the “KD” column(results shown in Table 20). Additionally, PN660 had siRNA knockdownactivities for all siRNAs tested that were comparable to PN643 and PN690indicating that the removal of the 9 most N-terminal residues of thePN73 peptide did not affect siRNAs mediated knockdown activity of thetargeted TNF-α mRNA. PN654 showed moderate knockdown activity for boththe A19S21 and 21/21 siRNAs but not for the LC20 siRNA (knockdownactivity is shown by “±” in knockdown activity column). However, thesiRNAs complexed with either PN708 or PN735 resulted in no observableknockdown activity for any of the siRNAs.

Example 15 Polynucleotide Delivery-Enhancing Polypeptide PN708

As described above, the cell-uptake assay determines the number of cellsthat receive Cy5-conjugated siRNA when complexed with a peptide. siRNAcell-uptake was assessed by flow cytometry (refer to Example 2 fordetails). Uptake was expressed as a percentage calculated by dividingthe number of cells containing Cy5-conjugated siRNA by the total numberof transfected and untransfected cells in culture. Mean FluorescenceIntensity (MFI) was measured by flow cytometry and determined the amountof Cy5-conjugated siRNA found within cells. The MFI value directlycorrelates with the amount of Cy5-conjugated siRNA within the cell,thus, a higher MFI value indicates a greater number of Cy5-conjugatedsiRNA within the cells.

In this example, PN643 (full-length PN73 minus a C-terminal label),PN690 (full-length PN73 with a C-terminal FITC-label) and PN708 (15-merderived by deletion of the 21 N-terminal residues of PN73) were testedat 5 μM, 10 μM, 20 μM and 40 μM. PN643 and PN690 were also tested at 2.5μM and PN690 was additionally tested at 1.25 μM. PN643 and PN708 werealso both tested at 80 μM.

As shown in Table 21, the non-FITC labeled PN73 (PN643) peptide achievednearly 100% uptake of siRNA at 10 μM concentration. However, when thePN73 peptide was labeled with the FITC tag (PN690), its maximumcell-uptake activity was reduced to approximately 70%. PN708 showed adose dependent increase in siRNA cell-uptake activity. PN708 achieved amaximum siRNA cell-uptake activity of 95% at 80 μM. For the full-lengthPN73 peptides, cell viability decreased as the concentration of peptideincreased. In contrast, cells incubated with the PN708 peptidemaintained over 90% cell viability in the presence of all testedconcentrations. In this example, the truncated peptide PN708 aboutdoubled the amount of Cy5-siRNA delivered into cells compared to thefull-length PN73 (PN690) peptide.

TABLE 21 Summary of siRNA Delivery-Enhancing Characteristics of PN708 %siRNA Peptide Cell- siRNA % Cell Treatment Concentration Uptake Cy5-MFIViability Negative Control 0 0  0 98% (no treatment) Cy5-LC20 siRNA +2.5 μM 61% 8 95% PN643 5 μM 96% 13 95% 10 μM 97% 17 94% 20 μM 84% 10 93%40 μM 44% 7 78% 80 μM 12% 14 26% Cy5-LC20 siRNA + 1.25 μM 30% 7 95%PN690 2.5 μM 47% 17 97% 5 μM 71% 56 94% 10 μM 64% 67 92% 20 μM 55% 9091% 40 μM 45% 218 71% Cy5-LC20 siRNA + 5 μM 35% 9 96% PN708 10 μM 55% 2396% 20 μM 83% 85 97% 20 μM 93% 212 94% 80 μM 96% 378 91%

Polypeptide PN708 was characterized by determining its affect on siRNAmediated target gene expression reduction. The C-terminal FITC-label ofthe PN708 peptide was removed prior to assessing its ability to enhancetargeted gene expression reduction when complexed with a siRNA. In theabsence of the FITC-label, the truncated exemplary polynucleotidedelivery-enhancing polypeptide was named PN766 (refer to Table 19 inExample 12). The ability of siRNA/peptide complexes to modulateexpression of the human tumor necrosis factor-α (hTNF-α) gene wasassessed (protocol details can be found in Example 3). In this example,the random siRNA sequence, Qneg, served as a negative control and thesiRNAs LC20 and LC17 were used to target the hTNF-α mRNA in humanmonocytes. The molar ratios of siRNA to peptide tested were 1:5; 1:10;1:25; 1:50; 1:75 and 1:100. Both LC20 and LC17 were used at 20 nMconcentration.

The knockdown results were that both the LC20/PN766 and LC17/PN766siRNA/peptide complexes at 1:5; 1:10; and 1:25 reduced hTNF-α mRNAlevels to approximately 70%-80% of the Qneg siRNA negative control(i.e., 20%-30% reduction in mRNA levels compared to the Qneg negativecontrol). The siRNA/peptide ratios of 1:50; 1:75 and 1:100 had nosignificant affect on hTNF-α mRNA levels compared to the Qneg control.No cytotoxicity effects were observed with human monocytes in thepresence of the PN766 peptide.

Example 16 Peptide Mediated siRNA Cell-Uptake Activity

The siRNA cell-uptake assay and MFI measurements were performed asdescribed previously in Examples 2 and 3. The data is summarized inTable 22. Each peptide was tested at 0.63 μM, 1.25 μM, 2.5 μM and 5 μMconcentrations.

TABLE 22 Summary of PN73 Mutant Mediated siRNA Delivery Characteristics% siRNA Cell- siRNA % Cell Peptide Concentration Uptake Cy5 MFIViability No Treatment N/A 0% 2 92% PN73 0.63 μM 52% 2 87% 1.25 μM 62% 482%  2.5 μM 74% 14 88%   5 μM 91% 22 93% PN644 0.63 μM 67% 4 88% 1.25 μM71% 8 90%  2.5 μM 70% 24 86%   5 μM 83% 37 87% PN645 0.63 μM 68% 5 84%1.25 μM 70% 11 89%  2.5 μM 78% 21 90%   5 μM 88% 28 90% PN646 0.63 μM67% 4 81% 1.25 μM 70% 10 85%  2.5 μM 73% 24 87%   5 μM 88% 24 92% PN6470.63 μM 71% 13 85% 1.25 μM 74% 34 83%  2.5 μM 83% 39 88%   5 μM 85% 4187% PN729 0.63 μM 61% 4 82% 1.25 μM 69% 10 91%  2.5 μM 79% 16 92%   5 μM86% 30 90%

Example 17 Polynucleotide Delivery-Enhancing Polypeptides

Polynucleotide delivery-enhancing polypeptides shown in Table 23 werescreened for their ability to deliver siRNA into mouse tail fibroblast(MTF) cells.

TABLE 23 Delivery-Enhancing Polypeptides Screened for siRNA Cell-UptakeActivity Peptide Amino Acid Sequence Name PN680 (SEQ ID NO: 87)Androctonin RSVCRQIKICRRRGGCYYKCTNRPY-amide PN665 (SEQ ID NO: 88)Paradaxin GFFALIPKIISSPLFKTLLSAVGSALSSSGDQE-amide PN734 (SEQ ID NO: 89)m-Calpain + GTAMRILGGVIPRKKRRQRRRPPQ-amide TAT PN681 (SEQ ID NO: 90)MARCKS KKKKKRFSFKKSFKLSGFSFKKNKK-amide PN694 (SEQ ID NO: 91) PenetratinRQIKIWFQNRRMKWKK-amide PN714 (SEQ ID NO: 92) PenArgRQIRIWFQNRRMRWRR-amide PN760 (SEQ ID NO: 93) TAT + PeptideRKKRRQRRRPPVAYISRGGVSTYYSDTVKGRFTRQKYNKRA- P3a amide PN759 (SEQ ID NO:94) Bindin + TAT LGLLLRHLRHHSNLLANIPRKKRRQRRRPP-amide PN682 (SEQ ID NO:95) Pep-1 KETWWETWWTEWSQPKKKRKV-amide

The siRNA cell-uptake activity for the polynucleotide delivery-enhancingpolypeptides listed in Table 23 complexed with siRNA. Table 24summarizes the siRNA cell-uptake data, mean fluorescence intensity (MFI)measurements and cell viability data for each of the polypeptides.Polypeptides that achieved a percent siRNA cell-uptake of 75% or greaterare highlighted in grey in the “Treatment” column. The specific percentsiRNA cell-uptake for each these highlighted siRNA/peptide complexes isalso highlighted in grey in the “% siRNA Cell-Uptake” column.

LC20 is an oligo used for the siRNA targeting of the human tumornecrosis factor-alpha (hTNF-α) mRNA and is represented by theribonucleotide sequence:

UAGGGUCGGAACCCAAGCUUA (SEQ ID NO: 96)

siRNA uptake by cells was assessed by flow cytometry (refer to Example 2for details). Uptake was expressed as a percentage calculated bydividing the number of cells containing Cy5-conjugated siRNA by thetotal number of transfected and untransfected cells in culture. MeanFluorescence Intensity (MFI) was measured by flow cytometry anddetermined the amount of Cy5-conjugated siRNA found within cells. TheMFI value directly correlates with the amount of Cy5-conjugated siRNAwithin the cell, thus, a higher MFI value indicates a greater number ofCy5-conjugated siRNA within the cells.

The data show that PN680, PN681, PN709, PN760, PN759, and PN682, whencomplexed with siRNA, deliver siRNA into cells. The results for thescreening of polypeptides shown in Table 23 are shown in Table 24.

TABLE 24 Data of Polypeptide Mediated siRNA Delivery Screen (NT = nottested) Treatment % siRNA Cy5- siRNA/peptide Peptide Cell- siRNA % CellComplex Concentration Uptake MFI Viability No treatment N/A  0.0% 0.097.6% Cy5-LC20 + PN643 5 μM 95.4% 7.2 98.8% (positive control)Cy5-LC20 + PN680 0.63 μM  0.2% N/T 98.2% 2.5 μM  1.8% 1.4 98.3% 10 μM82.6% 4.5 99.2% 40 μM 79.1% 5.2 95.7% Cy5-LC20 + PN665 0.63 μM  0.0% N/T97.7% 2.5 μM  0.6% N/T 95.1% 10 μM N/T N/T N/T 40 μM N/T N/T N/TCy5-LC20 + PN734 0.63 μM  0.1% N/T 98.2% 2.5 μM  0.2% N/T 98.7% 10 μM 1.2% 1.3 98.4% 40 μM  4.5% 1.6 97.0% Cy5-LC20 + PN681 0.63 μM  0.2% 1.897.1% 2.5 μM 69.9% 4.6 98.9% 10 μM 97.3% 15.3 98.2% 40 μM 91.2% 13.792.6% Cy5-LC20 + PN694 0.63 μM  0.2% 1.4 97.1% 2.5 μM  0.2% 1.8 97.9% 10μM 48.0% 4.2 97.8% 40 μM 54.0% 3.9 83.6% Cy5-LC20 + PN714 0.63 μM  0.4%1.2 95.1% 2.5 μM  0.5% 2.3 96.4% 10 μM 19.1% 2.5 97.6% 40 μM 43.0% 4.994.7% Cy5-LC20 + PN709 0.63 μM  0.1% 1.0 94.0% 2.5 μM  0.2% 1.0 96.6% 10μM 18.6% 1.9 97.1% 40 μM 76.6% 5.8 97.1% Cy5-LC20 + PN760 0.63 μM   60%2.9 84.7% 2.5 μM 85.5% 78.5 90.8% 10 μM 90.6% 96.9 91.9% 40 μM 82.8%77.4 83.2% Cy5-LC20 + PN759 0.63 μM   43% 2.1 81.7% 2.5 μM 72.9% 7.385.2% 10 μM 83.6% 40.9 86.7% 40 μM   25% 10.5 26.6% Cy5-LC20 + PN6820.63 μM 52.1% 2.4 86.2% 2.5 μM 50.6% 2.2 91.3% 10 μM 56.9% 2.3 90.6% 40μM   92% 9.0 97.1%

As shown in the column entitled “% siRNA Cell-Uptake” of Table 24, the“no treatment” negative control showed no siRNA cell-uptake while thepositive control peptide achieved a percent siRNA cell-uptake activityof 95%. The Cy5 conjugated LC20 siRNA complexed with the polynucleotidedelivery-enhancing polypeptides PN680; PN681; PN709; PN760; PN759 orPN682 achieved a percent siRNA cell-uptake activity that exceeded 75% orgreater. The polypeptides PN694 and PN714 exhibited a moderate siRNAcell-uptake activity of 54% and 43%, respectively. The polypeptidesPN665 and PN734 demonstrated no significant siRNA cell-uptake activity(less than 5%).

The polypeptides were further characterized for their ability totransfect siRNAs into cells by analyzing Mean Fluorescence Intensity(MFI). While the cell-uptake assay determined the percentage of cellsthat contain the Cy5-conjugated siRNA, the MFI measurement determinedthe relative mean quantity of Cy5-conjugated siRNA that entered thecells. As shown in the column entitled “siRNA Cy5 MFI” of Table 24,delivery of the Cy5-conjugated siRNA by the positive control peptidePN643 achieved a MFI of approximately seven units. As expected, the “notreatment” negative control has no measurable MFI. The polynucleotidedelivery-enhancing polypeptide PN665 was not tested by MFI. PN743, PN694and PN714 had MFI measurements significantly lower than that of thepositive control. The polynucleotide delivery-enhancing polypeptidesPN680, PN709 and PN682 exhibited MFI measurements comparable to that ofthe PN643 positive control while PN681 had an MFI double that of thepositive control. PN760 and PN759 had MFI measurements that wereapproximately 13-fold and 6-fold greater, respectively, than that of thepositive control.

The following protocol was used to test the polynucleotidedelivery-enhancing polypeptides listed in Table 23. Approximately 80,000mouse tail fibroblast (MTF) cells were plated per well in 24-well platesthe day before transfection in complete media. Each delivery peptide,except the positive control, was tested at 0.63 μM, 2.5 μM, 10 μM and 40μM concentrations in the presence of 0.5 μM Cy5-conjugated siRNA. ForsiRNA/peptide complexes, the Cy5-conjugated siRNA and peptide werediluted separately in Opti-MEM® media (Invitrogen) at two-fold the finalconcentration. Equal volumes of siRNA and peptide were mixed and allowedto complex five minutes at room temperature. The siRNA/peptide complexeswere added to cells previously washed with phosphate buffered saline(PBS). Cells were transfected for three hours at 37° C., 5% CO₂. Cellswere washed with PBS, treated with trypsin, and then analyzed by flowcytometry. siRNA cell-uptake was measured by the intensity ofintracellular Cy5 fluorescence. Cell viability was determined usingpropidium iodide uptake or AnnexinV-PE (BD Biosciences) staining. Inorder to differentiate the cellular uptake from the membrane insertionof labeled siRNA (or fluorescein-labeled peptide), trypan blue was usedto quench any fluorescence on the cell membrane surface. Trypan blue(Sigma) was added to cells to a final concentration of 0.04% and re-runon the flow cytometer to assess whether there was any change influorescence intensity which would indicate fluorescence localized tothe cell membrane.

Example 18 Knockdown Activity of siRNAs with Polypeptides

The ability of siRNA/peptide complexes to modulate expression of thehuman tumor necrosis factor-α (hTNF-α) gene was assessed.

Human monocytes were used as a model system to determine the effect ofsiRNA/peptide complexes on hTNF-α gene expression. Qneg represents arandom siRNA sequence and functioned as the negative control. Theobserved Qneg knockdown activity was normalized to 100% (100% geneexpression levels) and the knockdown activity for each of the followingsiRNAs A19S21 MD8, 21/21 MD8 and LC20 was presented as a relativepercentage of the negative control. A19S21 MD8, 21/21 MD8 and LC20 aresiRNAs that target hTNF-α mRNA.

The polypeptide PN602 is an acetylated form of the positive control usedin prior Examples and was used in this example as a positive control forboth the effective delivery of siRNA into human monocytes and thepermissive knockdown activity of hTNF-α mRNA levels mediated by siRNA.

The data show that the polynucleotide delivery-enhancing polypeptidePN680 delivers siRNAs into cells and permits effective siRNA mediatedgene silencing. The knockdown activity of PN602, PN680, and PN681 isshown in Table 25. A “+” symbol indicates that the peptide/siRNA complexhad knockdown activity of 80% of the Qneg negative control siRNA (20%reduction in mRNA levels compared to the Qneg negative control). A “+/−”indicates that the peptide/siRNA complex had a knockdown activity ofapproximately 90% of the Qneg negative control siRNA (10% reduction inmRNA levels compared to the Qneg negative control). Finally, a “−”indicates that the peptide/siRNA complex had no significant knockdownactivity compared to the Qneg negative control.

TABLE 25 siRNA Knockdown Activity for siRNAs Complexed With PolypeptidessiRNA siRNA:Peptide A19S21 Peptide ID # Ratio MD8 21/21 MD8 LC20 PN6021:5  +/− +/− +/− (Positive control) 1:10 +/− +/− +/− PN680 1:5  + + +1:10 +/− +/− + PN681 1:5  +/− − − 1:10 +/− − −

The results shown in Table 25 indicate that all three siRNAs complexedwith the positive control PN602 polynucleotide delivery-enhancingpolypeptide at ratios of 1:5 and 1:10 moderately reduced hTNF-α geneexpression levels compared to the Qneg negative control complexed withthe same polypeptide. However, the same siRNAs complexed with thepolynucleotide delivery-enhancing polypeptide PN681 at 1:5 and 1:10showed little to no knockdown activity relative to the Qneg negativecontrol siRNA/PN681 complex. In contrast, the polynucleotidedelivery-enhancing polypeptide PN680 complexed with any of the hTNF-αspecific siRNAs at a 1:5 ratio exhibited significant knockdown activityof the hTNF-α mRNA relative to the Qneg/PN680 control complex.Furthermore, the LC20/PN680 complex at a 1:10 ratio also demonstratedsignificant knockdown activity compared to the Qneg/PN680 controlcomplex.

Healthy human blood was purchased from Golden West Biologicals (CA), theperipheral blood mononuclear cells (PBMC) were purified from the bloodusing Ficoll-Pague plus (Amersham) gradient. Human monocytes were thenpurified from the PBMCs fraction using magnetic microbeads from MiltenyiBiotech. Isolated human monocytes were resuspended in IMDM supplementedwith 4 mM glutamine, 10% FBS, 1× non-essential amino acid and 1×pen-strep, and stored at 4 C until use.

In a 96 well flat bottom plate, human monocytes were seeded at100K/well/100 μl in OptiMEM medium (Invitrogen). The polynucleotidedelivery-enhancing polypeptides were mixed with 20 nM siRNA at a molarratio of 1:5 or 1:10 in OptiMEM medium at room temperature for fiveminutes. At the end of incubation, FBS was added to the mixture (final3%), and 50 μl of the mixture was added to the cells. The cells wereincubated at 37° C. for 3 hours. After incubation, the cells weretransferred to V-bottom plate and pelleted at 1500 rpm for five minutes.The cells were resuspended in growth medium (IMDM with glutamine,non-essential amino acid, and pen-strep). After an overnight incubation,the monocytes were stimulated by application of LPS (Sigma) at 1 ng/mlfor three hours to increase expression of TNF-α expression levels. Afterinduction by LPS, cells were collected as above for mRNA quantification,and supernatant was saved for protein quantification if desired.

For mRNA measurement, branch DNA technology from Genospectra (CA) wasused according to manufacturer's specification. To quantitate mRNA levelin the cells, both house keeping gene (cypB) and target gene (TNF-α)mRNA were measured, and the reading for TNF-α was normalized with cypBto obtain relative luminescence unit.

1-60. (canceled)
 61. A composition comprising a double-stranded RNA, afirst peptide and a second peptide, wherein the concentration of thefirst peptide of the composition provides an N/P ratio of the firstpeptide and the dsRNA of from about 0.2 to about 1, and wherein theconcentration of the second peptide of the composition provides an N/Pratio of the composition from about 0.5 to about 20, and wherein thedsRNA, the first peptide and the second peptide form a particle having adiameter of from about 0.5 nm to about 1000 nm.
 62. The composition ofclaim 61, wherein the first peptide is from 5% to 99% of the mass of theparticle.
 63. The composition of claim 61, wherein the first peptide isfrom 5% to 50% of the mass of the particle.
 64. The composition of claim61, wherein the first peptide and the second peptide are from 50% to 99%of the mass of the particle.
 65. The composition of claim 61, whereinthe amino acid sequence of the first peptide and the second peptideindependently comprise an amino acid sequence selected from the groupconsisting of SEQ ID NOS:28-37, 43, 67-71, and 87-95.
 66. Thecomposition of claim 61, wherein the amino acid sequence of the firstpeptide and the second peptide independently comprise an amino acidsequence selected from the group consisting of SEQ ID NO:28, 34, 37, 38,and
 39. 67. The composition of claim 61, wherein the amino acid of thefirst peptide comprises the amino acid sequence of SEQ ID NO:
 28. 68.The composition of claim 61, wherein the first peptide and/or the secondpeptide is pegylated.
 69. The composition of claim 61, wherein theparticle has a diameter of from 10 to 300 nanometers.
 70. Thecomposition of claim 61, wherein the particle has a diameter of from 40to 100 nanometers.
 71. The composition of claim 61, wherein the particleis cross-linked.
 72. The composition of claim 61, wherein the particlehas a zeta potential magnitude of at least 20 mV.
 73. The composition ofclaim 61, wherein the particle has a zeta potential magnitude of atleast 30 mV
 74. The composition of claim 61, wherein the dsRNA is ansiNA.
 75. A method comprising the steps of: a) associating a firstpeptide and a double-stranded RNA (dsRNA), wherein the first peptide andthe dsRNA form a first particle and have an N/P ratio of from about 0.2to about 1; b) associating a second peptide with the first particle,wherein the second peptide adjusts the N/P ratio to about 0.5 to about20; and wherein the first peptide, the second peptide, and the dsRNAform a second particle having diameter of less than 1000 nm.
 76. Themethod of claim 75, wherein the first peptide is from 5% to 99% of themass of the second particle.
 77. The method of claim 75, wherein thefirst peptide is from 5% to 50% of the mass of the second particle. 78.The method of claim 75, wherein the first peptide and the second peptideare from 50% to 99% of the mass of the second particle.
 79. The methodof claim 75, wherein the amino acid sequence of the first peptide andthe second peptide independently comprise an amino acid sequenceselected from the group consisting of SEQ ID NOS:28-37, 43, 67-71, and87-95.
 80. The method of claim 75, wherein the amino acid sequence ofthe first peptide and the second peptide independently comprise an aminoacid sequence selected from the group consisting of SEQ ID NO:28, 34,37, 38, and
 39. 81. The method of claim 75, wherein the amino acid ofthe first peptide comprises the amino acid sequence of SEQ ID NO: 28.82. The method of claim 75, wherein the first peptide and/or the secondpeptide is pegylated.
 83. The method of claim 75, wherein the secondparticle has a diameter of from 10 to 300 nanometers.
 84. The method ofclaim 75, wherein the second particle has a diameter of from 40 to 100nanometers.
 85. The method of claim 75, wherein the second particle hasa zeta potential magnitude of at least 20 mV.
 86. The method of claim75, wherein the second particle has a zeta potential magnitude of atleast 30 mV.
 87. The method of claim 75, wherein the second particle iscross-linked.
 88. The method of claim 75, wherein the first peptide ismixed or admixed with the dsRNA.
 89. The method of claim 75, wherein thesecond peptide is mixed or admixed with the first peptide and the dsRNA.